Administration of an anti-lgr5 monoclonal antibody

ABSTRACT

The present invention relates generally to the field of cancer biology. Some embodiments of the methods and compositions provided herein relate to administration of humanized antibodies or antigen-binding fragments thereof that specifically bind to LRG5 to treat certain cancers

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/311,631 entitled “ADMINISTRATION OF AN ANTI-LGR5 MONOCLONAL ANTIBODY” filed Mar. 22, 2016 the contents of which are expressly incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to the field of cancer biology. Some embodiments of the methods and compositions provided herein relate to administration of humanized antibodies or antigen-binding fragments thereof that specifically bind to leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) to treat certain cancers.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIONO14WOSEQLISTING, created Mar. 8, 2017 which is approximately 40 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5), also known as GPR49/HG38/FEX, belongs to the leucine-rich repeat containing G-protein-coupled receptor (LGR)/G-Protein-coupled Receptor (GPR) protein family of receptor proteins that are structurally similar to glycoprotein hormone receptors. LGRs are divided into three subgroups: (1) glycoprotein hormone receptors including thyroid-stimulating hormone (TSH) receptor, follicle-stimulating hormone (FSH) receptor, and luteinizing hormone (LH) receptor; (2) relaxin receptors LGR7 and LGR8; and (3) LRG4, LGR5, and LGR6. LGR5 is expressed in several tissues including the intestine, skeletal muscle, placenta, brain, and spinal cord.

SUMMARY OF THE INVENTION

Embodiments of the methods and compositions provided herein include a method of treating a human subject having a metastatic colorectal cancer comprising administering an effective amount of a humanized monoclonal antibody that specifically binds leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) to the subject in need thereof, wherein: the monoclonal antibody comprises a heavy chain comprising SEQ ID NO:13 and a light chain comprising SEQ ID NO:14; the monoclonal antibody is administered weekly for at least 4 weeks; the monoclonal antibody is administered intravenously; and the dosage of the monoclonal antibody is between about 2.5 mg/kg to about 15 mg/kg.

In some embodiments, the monoclonal antibody is administered in combination with folinic acid, fluorouracil, and irinotecan. In some embodiments, an initial dose of the monoclonal antibody is administered prior to administration of the folinic acid, fluorouracil, and irinotecan. In some embodiments, an initial dose of the irinotecan is about 180 mg/m2 administered over about 90 minutes; an initial dose of the folinic acid is about 400 mg/m2 administered over about 120 minutes and concurrently with the initial dose of the irinotecan; an initial dose of the fluorouracil is about 400 mg/m2 administered after administration of the initial dose of the folinic acid; and the folinic acid, fluorouracil, and irinotecan are administered every 14 days.

Embodiments of the methods and compositions provided herein include a method of treating a subject having a cancer comprising administering an effective amount of a humanized monoclonal antibody or an antigen-binding fragment thereof that specifically binds leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) to the subject in need thereof, wherein: the monoclonal antibody comprises a heavy chain comprising SEQ ID NO:13 and a light chain comprising SEQ ID NO:14; the monoclonal antibody is administered weekly for at least 4 weeks; the monoclonal antibody is administered intravenously; and the dosage of the monoclonal antibody is between about 2.5 mg/kg to about 15 mg/kg.

In some embodiments, the monoclonal antibody is administered in combination with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of folinic acid, fluorouracil, irinotecan, gemcitabine and nanoparticle albumin-bound paclitaxel (ABRAXANE).

In some embodiments, an initial dose of the monoclonal antibody is administered prior to administration of a chemotherapeutic agent.

In some embodiments, the monoclonal antibody is administered in combination folinic acid, fluorouracil, and irinotecan. In some embodiments, an initial dose of the monoclonal antibody is administered prior to administration of folinic acid, fluorouracil, and irinotecan.

In some embodiments, an initial dose of the irinotecan is about 180 mg/m2 administered over about 90 minutes. In some embodiments, an initial dose of the folinic acid is about 400 mg/m2 administered over about 120 minutes and concurrently with the initial dose of the irinotecan. In some embodiments, an initial dose of the fluorouracil is about 400 mg/m2 administered after administration of the initial dose of the folinic acid. In some embodiments, the folinic acid, fluorouracil, and irinotecan are administered every 14 days.

In some embodiments, the monoclonal antibody is administered in combination with an additional therapeutic agent selected from the group consisting of bevacizumab, aflibercept, cetuximab, and panitumumab.

In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, and lung cancer. In some embodiments, the cancer is selected from the group consisting of colon cancer comprising an APC mutation, colon cancer comprising an KRAS mutation, metastatic colorectal cancer, metastatic pancreatic cancer, triple-negative breast cancer, and small cell lung cancer. In some embodiments, the cancer is a metastatic colorectal cancer.

In some embodiments, the subject has a characteristic selected from the group consisting of: failed at least 1 line of prior chemotherapy for metastatic disease prior to administration of the monoclonal antibody; has no known brain metastases; has a life expectancy of 12 weeks or more; has an absolute neutrophil count greater than about 1500 cells/mL without growth factor support in the 14 days prior to administration of the monoclonal antibody; has a platelet count greater than 100,000 platelets/mL without transfusions in the 14 days prior to administration of the monoclonal antibody; has a hemoglobin greater than or equal to 9.0 g/dL; and has serum albumin greater than or equal to 3 g/dL.

In some embodiments, the subject is mammalian, for example, human.

Embodiments of the methods and compositions provided herein include a container comprising a pharmaceutical composition comprising a dose of a humanized monoclonal antibody that specifically binds leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), and a suitable pharmaceutical carrier, wherein the dose of the monoclonal antibody is between about 2.5 mg/kg to about 15 mg/kg. In some embodiments, the pharmaceutical composition is suitable for intravenous administration.

Embodiments of the methods and compositions provided herein include a humanized monoclonal antibody that specifically binds leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) for use in treating a metastatic colorectal cancer wherein: the monoclonal antibody comprises a heavy chain comprising SEQ ID NO:13 and a light chain comprising SEQ ID NO:14; the monoclonal antibody is administered weekly for at least 4 weeks; the monoclonal antibody is administered intravenously; and the dosage of the monoclonal antibody is between about 2.5 mg/kg to about 15 mg/kg. In some embodiments, the monoclonal antibody is administered in combination with folinic acid, fluorouracil, and irinotecan. In some embodiments, an initial dose of the monoclonal antibody is administered prior to administration of the folinic acid, fluorouracil, and irinotecan. In some embodiments, an initial dose of the irinotecan is about 180 mg/m2 administered over about 90 minutes; an initial dose of the folinic acid is about 400 mg/m2 administered over about 120 minutes and concurrently with the initial dose of the irinotecan; an initial dose of the fluorouracil is about 400 mg/m2 administered after administration of the initial dose of the folinic acid; and the folinic acid, fluorouracil, and irinotecan are administered every 14 days.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing direct FACS binding of humanized monoclonal antibody 18G7H6A3 to human LGR5 (CHO).

FIG. 2 is a graph showing the effect of FOLFIRI, alone and in combination with 18G7H6A3, on CT3 CRC tumor volume.

FIG. 3 is a graph showing 18G7H6A3 treatment significantly reduced MDA-MB-231-LM3 primary tumor volume.

FIG. 4 shows graphs of FolFiri treatment in mice bearing CT1, or CT3 tumors results in upregulation of LGR5.

FIG. 5 is a bar chart showing chemotherapy results in upregulation of LGR5 (more than 4-fold) in JH109 tumors.

FIG. 6 is a graph showing significant activity of 18G7H6A3 observed when administered in combination with chemotherapy (gemcitabine).

FIG. 7 is a point plot showing that antibody 18G7H6A3 reduces the number of live events in a CT1 cancer stem cell population.

FIG. 8 is a line graph showing cells isolated from mice treated with anti-LGR5 antibody 18G7H6A3 in combination with FOLFIRI had greatly decreased tumorigenicity as compared to cells isolated from mice treated with FOLFIRI alone.

FIG. 9 is a line graph showing that re-implanted cells from the 18G7H6A3 FOLFIRI combination had a significantly delayed time to progression.

FIG. 10 is a line graph showing significant activity of humanized antibody 18G7H6A3 is observed when administered prophylactically in combination with chemotherapy (FOLFIRI).

FIG. 11 is a point plot showing that antibody 18G7H6A3 is able to inhibit Wnt signaling in tumor cells in vivo as indicated by phospho-Thr41/Ser45-β-catenin immunoassays.

FIG. 12 is a bar chart showing that increasing concentrations of soluble antibody 18G7H6A3 did not affect the induction of TCF/LEF promoter driven GFP expression by the combination of Wnt3a plus RSPO2, demonstrating that the anti-LGR5 antibody 18G7H6A3 does not block RSPO-driven TCF/LEF promoter activation. A positive control antibody C12 is shown to inhibit Wnt3a/RSPO2 driven TCF/LEF promoter activitation.

FIG. 13 is a line graph showing that R-spondin does not block antibody 18G7H6A3 binding to LGR5.

FIG. 14 is a bar chart showing that antibody 18G7H6A3 binding to LGR5 inhibits formation of ternary complex.

FIG. 15 depicts levels of LGR5 expression in treated samples.

FIG. 16 depicts levels of CTNNB1 expression, and p-β-Catenin expression in treated samples.

FIG. 17 depicts differentially expressed transcripts in various treated samples.

FIG. 18 depicts differentially expressed genes in 18G7H6A3- (BNC101) treated tumors.

FIG. 19 depicts differentially expressed genes in FOLFIRI treated tumors.

FIG. 20 depicts differentially expressed genes in combination-treated tumors

FIG. 21 depicts levels of LGR5 in circulating HLA+ cells.

FIG. 22A and FIG. 22B depict levels of LGR5 in circulating HLA+ cells.

FIG. 23 is a graph showing animal survival of mice treated with Gemcitabine/Abraxane or with Gemcitabine/Abraxane and 18G7H6A3.

DETAILED DESCRIPTION

Some embodiments of the methods and compositions provided herein relate to administration of humanized antibodies or antigen-binding fragments thereof that specifically bind to leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) to treat certain cancers An embodiment of such humanized antibodies and antigen-binding fragments thereof is disclosed in PCT Publication No. WO 2015/153916 published Oct. 8, 2017 which is incorporated by reference in its entirety.

LGR5 was identified through lineage tracing studies as a highly specific marker of normal stem cells and tumor-initiating cells in the gut. Previously about 150 genes were identified whose expression was quenched following abrogation of Wnt expression. A comprehensive characterization of these ‘Wnt target genes’ found LGR5 to be selectively expressed on a population of 10-14 proliferating wedge-shaped cells at the crypt base. These crypt-based columnar cells were previously proposed to be a candidate stem cell population. Using in vivo lineage tracing with a heritable lacZ-LGR5 reporter gene, it has been confirmed that LGR5 intestinal stem cells are a multi-potent, self-renewing population of adult intestinal stem cells that give rise to uninterrupted ribbons of lacZ+ progeny cells initiating from the crypt base and extending to the villus tips.

The specific expression of LGR5 on cancer stem cells (CSCs) provides an opportunity to target CSCs selectively and effectively. LGR5 is highly over expressed in CRC, pancreatic and most other solid tumors, compared to normal tissues, thereby providing a wide therapeutic window to target CSCs in CRC, pancreatic, breast, ovarian, lung, gastric and liver cancer.

The gate keeping mutation in CRC is loss of adenomatous polyposis coli (APC), resulting in the aberrant activation of Wnt signaling, which normally acts to regulate the balance between stem cell self-renewal and differentiation in the colon crypt. Dysregulated Wnt signaling in intestinal stem cells leads to the formation of adenomatous polyps in the colon that are the precursor to malignant CRC. LGR5 stem cells were confirmed to be the source or root of these mouse intestinal tumors, using a strategy that crossed inducible APC gene knockout mice with mice whose LGR5 stem cells were specifically and randomly labeled with one of four (GFP/YFP/ECFP/RFP) fluorescent genetic markers. The appearance of single colored tumors (i.e., all GFP or all RFP) 4 weeks after induction of APC deletion confirmed that these tumors arose from a single LGR5 stem cell. Furthermore, this model also allowed for the fluorescent genetic tag in the LGR5 stem cells to be flipped to a different color, so that an RFP+ LGR5 cancer stem cell generating a red tumor could be transformed midstream into a ECFP+ LGR5 cancer stem cell, that was still seeding the tumor but now giving rise to blue tumor cells invading the previously all red GFP+ tumor mass. This flipping experiment not only provided further confirmation that LGR5 CSCs are the origin of intestinal tumors, able to initiate and seed the growth of intestinal tumors, but also that they continuously maintain tumor formation (i.e., have long-term repopulating ability).

A functional role of LGR5 in cancer has been validated through ribonucleic acid interference (RNAi) knockdown studies. Knockdown of LGR5 in a panel of CRC tumor cell lines significantly inhibited the growth of soft agar colonies in vitro, and also the growth of HCT116 colon tumor xenografts in vivo. LGR5 RNAi knockdown was subsequently shown to also reduce the growth of CSC colonies from patient-derived CRC tumor cells in vitro (data not shown). Finally, sorted LGR5+ PATIENT DERIVED XENOGRAFT CRC tumor cells were found to be highly tumorigenic in vivo compared to control LGR5− cells.

CSCs are believed to responsible for the high incidence of tumor recurrence in many cancer patients treated with surgery and standard of care chemotherapy. For example, CD44+ CSCs from breast cancer patients were found to be enriched following chemotherapy, and that high levels of CSCs correlated with poor clinical response to chemotherapy. Similarly, in metastatic CRC, LGR5 expression was upregulated in damaged liver following chemotherapy, suggesting that increased LGR5 CSCs in response to chemotherapy initiate and/or acerbate metastatic disease. Indeed, it has been found that LGR5 expression is significantly greater in metastatic sites compared to primary CRC tumors.

Anti-LGR5 Antibodies

As used herein, the term “antibody” includes, but is not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv), Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv) (including bi-specific sdFvs), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. The antibodies of several embodiments provided herein may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide or may be specific for both a polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992); each of which is incorporated herein by reference in its entirety.

As used herein, LGR5 includes, but is not limited to, human LGR5 including the polypeptide of NCBI Accession No. NP 003658.1, or fragments thereof, which is encoded by the coding nucleotide sequence within NM 003667.2, or fragments thereof. The amino acid sequence and entire entry of NCBI Accession No. NP_003658.1 and nucleotide sequence and entire entry of NM_003667.2 are fully incorporated by reference in their entireties. Examples of LGR5 fragments contemplated herein include the LGR5 ectodomain, transmembrane domain, or intracellular domain and portions thereof.

Several embodiments relate to a hybridoma that produces the light chain and/or the heavy chain of an anti-LGR5 antibody, including the anti-LGR5 antibodies designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below. In one aspect, the hybridoma produces the light chain and/or the heavy chain of a humanized or fully human monoclonal antibody such as that of 18G7H6A3 and 18G7H6A1 produced and described in the Examples below.

Some embodiments are drawn to a nucleic acid molecule encoding the light chain or the heavy chain of an anti-LGR5 antibody, including any one of the anti-LGR5 antibodies designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below. In some aspects, a nucleic acid molecule encodes the light chain or the heavy chain of a humanized or fully human monoclonal, such as antibody 18G7H6A3 and 18G7H6A1 produced and described in the Examples below.

Various embodiments are directed to a vector comprising a nucleic acid molecule or molecules encoding a light chain and/or a heavy chain of an anti-LGR5 antibody, including any one of the anti-LGR5 antibodies designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below.

In various embodiments, the glycosylation of the antibodies can be modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861; each of which is incorporated herein by reference in its entirety.

In several embodiments, the antibodies specifically bind a polypeptide comprising or consisting of a LGR5 polypeptide having at least 60% identity, or at least 70% identity, or at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, or at least at least 97% identity, or at least 99% identity, or 100% identity to the human LGR5 polypeptide of NCBI Accession Nos. NP_003658.1 (SEQ ID NO: 47) or fragments thereof. Such fragments can, for example, be at least about 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 contiguous or non-contiguous amino acids of the LGR5 polypeptide, or any number of contiguous or non-contiguous amino acids in between any of the aforementioned lengths.

In several embodiments, the antibody is antibody 18G7H6A3 and comprises a heavy chain amino acid sequence of SEQ ID NO: 13 and a DNA sequence of SEQ ID NO: 11. In some embodiments, the antibody is antibody 18G7H6A3 and has a heavy chain variable domain comprises SEQ ID NO: 19. In several embodiments, the antibody is antibody 18G7H6A3 and comprises a light chain sequence of SEQ ID NO: 14. In other embodiments, the antibody is antibody 18G7H6A3 and comprises a light chain variable domain of SEQ ID NO: 21.

In some embodiments the antibodies comprise a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to the sequence of the above sequences. In some embodiments the antibodies comprise a sequence that is 100% identical to the above antibody sequences over a span of 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, or 118 residues of the heavy chain, light chain, or variable domains of the above sequences.

In some embodiments the antibodies comprise a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to the antibody sequences. In some embodiments the antibodies comprise a sequence that is 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or 100% identical to the antibody sequences. In some embodiments the antibodies comprise a sequence that is 100% identical to the antibody sequences of over a span of 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, or 111 residues.

In some embodiments, an anti-LGR5 antibody provided herein comprises a heavy chain CDR1 comprising GYSFTAYW (SEQ ID NO:23), a heavy chain CDR2 comprising ILPGSDST (SEQ ID NO:2), and a heavy chain CDR3 comprising ARSGYYGSSQY (SEQ ID NO:3). In some embodiments, an anti-LGR5 antibody provided herein comprises a light chain CDR1 comprising ESVDSYGNSF (SEQ ID NO:4), a light chain CDR2 comprising LTS, and a light chain CDR3 comprising QQNAEDPRT (SEQ ID NO:33).

In some embodiments, an anti-LGR5 antibody provided herein comprises: (a) a heavy chain CDR1 comprising variants of the above sequences having 1, 2, 3, or 4 amino acid substitutions. The antibody may also have a heavy chain CDR2 having a variant comprising 1, 2, 3, or 4 amino acid substitutions. The antibody may also have a heavy chain CDR3 having a variant comprising 1, 2, 3, or 4 amino acid substitutions. In addition to these modifications of the heavy chain, the antibody may also have a light chain CDR1 having a variant comprising 1, 2, 3, or 4 amino acid substitutions. The antibody may also have a light chain CDR2 having a variant comprising 1, 2, 3, or 4 amino acid substitutions. The antibody may also have a light chain CDR3 having 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative amino acid substitutions.

In some embodiments, an anti-LGR5 antibody provided herein comprises an antibody which comprises a heavy chain variable region having at least 80% or 90% sequence identity to the sequences described herein in the attached sequence listing. The antibody may also have a light chain variable region having at least 80% or 90% sequence identity to the antibody sequences described herein.

The percent identity of two amino acid sequences (or two nucleic acid sequences) can be determined, for example, by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The amino acids or nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions×100). The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A specific, non-limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.2) as described in Schaffer et al., Nucleic Acids Res., 29:2994-3005 (2001), which is incorporated herein by reference in its entirety. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN) can be used. See http://www.ncbi.nlm.nih.gov, as available on Apr. 10, 2002. In one embodiment, the database searched is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1.

Several embodiments also encompass variants of the above described antibodies, including any one of the anti-LGR5 antibodies designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below, comprising one or more amino acid residue substitutions in the variable light (V_(L)) domain and/or variable heavy (V_(H)) domain. Several also encompass variants of the above described antibodies with one or more additional amino acid residue substitutions in one or more V_(L) CDRs and/or one or more V_(H) CDRs. The antibody generated by introducing substitutions in the V_(H) domain, V_(H) CDRs, V_(L) domain and/or V_(L) CDRs of the above described antibodies can be tested in vitro and in vivo, for example, for its ability to bind to LGR5 (by, e.g., immunoassays including, but not limited to ELISAs and BIAcore).

Various embodiments include antibodies that specifically bind to LGR5 comprising derivatives of the V_(H) domains, V_(H) CDRs, V_(L) domains, or V_(L) CDRs of anti-LGR5 antibodies, such as any one of the anti-LGR5 antibodies designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below, that specifically bind to LGR5. Standard techniques known to those of skill in the art can be used to introduce mutations (e.g., additions, deletions, and/or substitutions) in the nucleotide sequence encoding an antibody, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis are routinely used to generate amino acid substitutions. In one embodiment, the V_(H) and/or V_(L) CDRs derivatives include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original V_(H) and/or V_(L) CDRs. In another embodiment, the V_(H) and/or V_(L) CDRs derivatives have conservative amino acid substitutions (e.g. supra) made at one or more predicted non-essential amino acid residues (i.e., amino acid residues which are not critical for the antibody to specifically bind to LGR5). Alternatively, mutations can be introduced randomly along all or part of the Vu and/or V_(L) CDR coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined.

Several embodiments also encompass antibodies that specifically bind to LGR5 or a fragment thereof, the antibodies comprising an amino acid sequence of a variable heavy chain and/or variable light chain that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the variable heavy chain and/or light chain of any of the antibodies described herein including any one of the anti-LGR5 antibodies including those designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below.

Another embodiment includes the introduction of conservative amino acid substitutions in any portion of an anti-LGR5 antibody, such as any one of the anti-LGR5 antibodies designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below. It is well known in the art that “conservative amino acid substitution” refers to amino acid substitutions that substitute functionally-equivalent amino acids. Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide. For example, one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide. Substitutions that are charge neutral and which replace a residue with a smaller residue may also be considered “conservative substitutions” even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine). Families of amino acid residues having similar side chains have been defined in the art. Several families of conservative amino acid substitutions are shown in Table 1.

TABLE 1 Family Amino Acids non-polar Trp, Phe, Met, Leu, Ile, Val, Ala, Pro uncharged polar Gly, Ser, Thr, Asn, Gln, Tyr, Cys acidic/negatively charged Asp, Glu basic/positively charged Arg, Lys, His Beta-branched Thr, Val, Ile residues that influence Gly, Pro chain orientation aromatic Trp, Tyr, Phe, His Blocking Cancer Stem Cell Growth with Anti-LGR5 Antibodies

Several embodiments are drawn to blocking cancer stem cell growth in vitro and in vivo with anti-LGR5 antibodies. In some embodiments, a method of blocking cancer stem cell growth comprises administering an effective amount of an anti-LGR5 antibody to cancer stem cells, wherein the effective amount of the anti-LGR5 antibody is sufficient to reduce growth of the cancer stem cells.

In some embodiments, a method of blocking cancer stem cell growth comprises administering an effective amount of an anti-LGR5 antibody to cancer stem cells, wherein the effective amount of the anti-LGR5 antibody is sufficient to reduce or block proliferation, or reduce or block the growth, of the cancer stem cells.

In some aspects, an effective amount of an anti-LGR5 antibody is administered to cancer stem cells in vitro. In other aspects, an effective amount of an anti-LGR5 antibody is administered to cancer stem cells in a patient in need of treatment thereof, in vivo.

As used herein, the term “cancer stem cell(s)” refers to a cell that can proliferate extensively or indefinitely and give rise to a large proportion of cancer cells in a cancer. In some aspects, the large proportion of cancer cells represents a majority of the cancer cells in a given cancer. For illustration, but not limitation, a cancer stem cell(s) can be a founder of a tumor or a progenitor of the cancer cells that comprise the majority of a cancer's mass. In some aspects, cancer stem cells refer to cells that divide to form one or more tumors when implanted into an immunocompromised individual, in the absence of any additional mutation to the cells or introduction of exogenous cell proliferation-inducing or carcinogenic agents. In some aspects cancer stem cells divide to yield additional cancer stem cells as well as terminally differentiated cancer cells or cancer tissue.

In some embodiments cancer stem cell growth, proliferation, or viability is blocked without interfering with LGR5-RSpo binding or signaling. In some embodiments cancer stem cell growth, proliferation, or viability is blocked without interfering with LGR5-RSpo binding or signaling through blocking or inhibiting LGR5 signaling through Wnt.

As used with respect to blocking cancer stem cell growth, the term “effective amount” refers to an amount of anti-LGR5 antibody sufficient to reduce the growth of cancer stem cells by any degree. Any assay known in the art can be used to measure cancer stem cell growth. For example, cancer stem cell growth can be measured by colony count, total cell count, or volume/size of a cell population or colony. In several embodiments, cancer stem cell growth can be measured by the tumor sphere growth assay described below in Example 1.

In certain embodiments, an effective amount of an anti-LGR5 antibody can block cancer stem cell growth as measured by at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% reduction in the cancer stem cell population or tumorsphere growth, or any percentage in between any of the aforementioned numbers. In some aspects, the anti-LGR5 antibody is any one or combination of the anti-LGR5 antibodies designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below.

For example, in some embodiments, an effective amount of an anti-LGR5 antibody can block cancer stem cell growth as measured by at least about 5%-99%, a 5%-80%, a 5 to 40%, a 10% to 99%, a 10 to 80%, a 10-60%, a 10%-40%, a 20 to 99%, a 20%-80%, a 20%-60%, a 20%-40%, a 50%-98%, 50%-80%, or a 60%-99% reduction in the cancer stem cell population or tumorsphere growth. In some aspects, the anti-LGR5 antibody is any one or combination of the anti-LGR5 antibodies designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below.

In other embodiments, the effective amount of an anti-LGR5 antibody can block cancer stem cell growth as measured by at least about a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 10, 25, 50, 75, 100, 200, or 1000-fold reduction in the cancer stem cell population or tumorsphere growth, or any fold-reduction in between any of the aforementioned numbers. In some aspects, the anti-LGR5 antibody is any one or combination of the anti-LGR5 antibodies designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below.

In some embodiments, the effective amount of an anti-LGR5 antibody sufficient to block cancer stem cell growth by any degree described above is in a concentration of about 1 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 500 nM, 550 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 500 μM, 550 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 75 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, 1 M, 5 M, 10 M, 15 M, 20 M, 25 M, 30 M, 35 M, 40 M, 45 M, 50 M, 75 M, 100 M, or any number in between any two of the aforementioned concentrations. In some aspects, an anti-LGR5 antibody composition may comprise both of antibodies designated as 18G7H6A3 and 18G7H6A1 produced and described in the Examples below.

In some embodiments, an anti-LGR5 antibody provided herein binds human LGR5 with a KD of less than about 200 nM, less than about 100 nM, less than about 80 nM, less than about 50 nM, less than about 20 nM, less than about 10 nM, less than about 1 nM, and a range between any of the foregoing values. In some embodiments, an anti-LGR5 antibody provided herein binds LGR5 with an affinity less than about 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, and within a range of any of the foregoing values. In some embodiments, an anti-LGR5 antibody provided herein binds LGR5 with an affinity greater than about 0.0001 nM, 0.001 nM, 0.01 nM, and within a range of any of the foregoing values.

In some embodiments, an anti-LGR5 antibody provided herein binds to an epitope comprising or consisting of or within amino acids T175, E176, Q180, R183, S186, A187, Q189, D247, E248, T251, R254, 5257, N258, K260 of SEQ ID NO: 47. In some embodiments, an anti-LGR5 antibody provided herein binds to an epitope comprising or consisting of or within leucine rich repeats 6-9 (See e.g., Chen et al. Genes Dev. 27(12):1345-50 which is incorporated by reference in its entirety). In some embodiments, an anti-LGR5 antibody provided herein binds to an epitope comprising or consisting of or within the convex surface of the LGR5 ecto domain (See e.g., Chen et al. Genes Dev. 27(12):1345-50 which is incorporated by reference in its entirety).

Some embodiments include methods of treating cancer comprising administering a therapeutically effective amount of an anti-LGR5 antibody provided herein to a subject in need thereof. In some embodiments, the cancer is selected from pancreatic cancer, colorectal cancer, lung cancer, pancreatic cancer, and breast cancer, such as triple negative breast cancer. In some embodiments, the colorectal cancer comprises an inactivating mutation in the adenomatous polyposis coli (APC) gene, does not comprise an inactivating mutation in the APC gene, or comprises a wild-type APC gene. In some embodiments, the cancer is. In some embodiments, the cancer comprises elevated levels of LGR5 protein. In some embodiments, the cancer is colon cancer that expresses elevated levels of LGR5. In some embodiments, the cancer is a pancreatic cancer that expresses elevated levels of LGR5, In some embodiments, the cancer is a breast cancer that expresses elevated levels of LGR5.

Some embodiments include methods of treating a disease in a subject wherein the disease is associated with activation of β-catenin, and/or aberrant β-catenin signaling. Some embodiments include administering a therapeutically effective amount of an anti-LGR5 antibody provided herein to a subject in need thereof.

Some embodiments include methods of treating a disease comprising administering a therapeutically effective amount of an anti-LGR5 antibody provided herein to a subject in need thereof in combination with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent. In some embodiments, the additional therapeutic agent comprises a biologic agent. Some embodiments include administering an anti-LGR5 antibody provided herein in combination with a chemotherapeutic agent and a biologic agent. In some embodiments, administering an anti-LGR5 antibody provided herein in combination with a chemotherapeutic agent can increase the expression level of LGR5 in a cancer, such as a tumor. Some embodiments of the methods provided herein include determining the level of LGR5 protein expression in a tumor or cancer.

Some embodiments of the methods provided herein include identifying a subject for treatment with an anti-LGR5 antibody provided herein. Some embodiments include determining if the subject has a tumor comprising an elevated expression level of LGR5 as compared to the expression of the same LGR5 protein in normal tissue. Some embodiments include selecting a subject for treatment if the tumor has an elevated level of LGR5 expression. Some embodiments also include determining if the subject has a tumor that comprises an inactivating mutation in the APC gene. Some embodiments also include selecting a subject for treatment if the tumor comprises an inactivating mutation in the APC gene.

Methods, compositions and related disclosure relevant to the above are provided in, for example, PCT Publication No. WO 2013/067055, published May 10, 2013, the contents of which are hereby incorporated by reference in their entirety, as well as for example, PCT Publication No. WO 2013/067054, published May 10, 2013, the contents of which are hereby incorporated by reference in their entirety, as well as for example, PCT Publication No. WO 2013/067057, published May 10, 2013, the contents of which are hereby incorporated by reference in their entirety, as well as for example, PCT Publication No. WO 2013/067060, published May 10, 2013, the contents of which are hereby incorporated by reference in their entirety.

Pharmaceutical Compositions

The humanized monoclonal antibody or an antigen-binding fragment thereof that specifically binds LGR5 provided herein, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the humanized monoclonal antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Kits

Some embodiments provided herein include kits. In some embodiments, a kit can include a humanized antibody provided herein. In some embodiments, the antibody is lyophilized. In some embodiments, the antibody is in aqueous solution. In some embodiments, the kit includes a pharmaceutical carrier for administration of the antibody. In some embodiments, the kit also includes a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is selected from folinic acid, fluorouracil, irinotecan, gemcitabine and nanoparticle albumin-bound paclitaxel (ABRAXANE).

Some embodiments include a container comprising a pharmaceutical composition comprising a dose of humanized monoclonal antibody or an antigen-binding fragment thereof that specifically binds LGR5, and a suitable pharmaceutical carrier, in which the dose is suitable to treat a subject having a cancer. The dose of the humanized monoclonal antibody or an antigen-binding fragment can be greater than, less than or equal to about 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, or a range between any two of the foregoing dosages. In some embodiments, the dose of the humanized monoclonal antibody or an antigen-binding fragment thereof is between about 2.5 mg/kg to about 20 mg/kg, or between about 2.5 mg/kg to about 15 mg/kg. In some embodiments, the pharmaceutical composition is suitable for intravenous administration. In some embodiments, the pharmaceutical composition is suitable for intraperitoneal injection.

Methods of Treatment

Some embodiments of the methods, compositions and kits include methods of treating a subject having a cancer. Some such methods include administering an effective amount of a humanized monoclonal antibody or an antigen-binding fragment thereof that specifically binds LGR5 to a subject in need thereof. The subject can be mammalian, for example, human.

In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer can be a colon cancer, colorectal cancer, a pancreatic cancer, a breast cancer, or a lung cancer. In some embodiments, the cancer can be a colon cancer comprising an APC mutation, a colon cancer comprising an KRAS mutation, a metastatic colorectal cancer, a metastatic pancreatic cancer, a triple-negative breast cancer, or a small cell lung cancer.

The humanized monoclonal antibody or an antigen-binding fragment thereof that specifically binds LGR5 can include a heavy chain CDR, such a heavy chain CDR1 comprising SEQ ID NO:23, a heavy chain CDR2 comprising SEQ ID NO:25, and/or a heavy chain CDR3 comprising SEQ ID NO:27. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can include a heavy chain variable domain comprising SEQ ID NO:19. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can include a heavy chain comprising SEQ ID NO:13. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can include a light chain CDR, such as a light chain CDR1 comprising SEQ ID NO:29, a light chain CDR2 comprising SEQ ID NO:31, and/or a light chain CDR3 comprising SEQ ID NO:33. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can include a light chain variable domain comprising SEQ ID NO:21. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can include a light chain comprising SEQ ID NO:14. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can include a heavy chain comprising SEQ ID NO:13 and a light chain comprising SEQ ID NO:14. In some embodiments, the humanized monoclonal antibody or an antigen-binding fragment thereof is 18G7H6A3.

The dosage of the humanized monoclonal antibody or an antigen-binding fragment thereof to treat a subject having a cancer can be greater than, less than or equal to about 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, or a range between any two of the foregoing dosages. In some embodiments, the dosage of the humanized monoclonal antibody or an antigen-binding fragment thereof is between about 2.5 mg/kg to about 20 mg/kg, or between about 2.5 mg/kg to about 15 mg/kg.

The frequency of administration of the humanized monoclonal antibody or an antigen-binding fragment thereof can be daily, weekly, or monthly. In some embodiments, administration can be once a day, every 2 days, every 3 days, every 4 days, every 5 days, or every 6 days. In some embodiments, administration can be once a week, every 2 weeks, every 3 weeks, or every 4 weeks. In some embodiments, administration can be monthly.

The route of administration of the humanized monoclonal antibody or an antigen-binding fragment thereof can be suitable for administration of a biologic. For example, administration can be via an intraperitoneal injection, or via an intravenous route.

Administration of the humanized monoclonal antibody or an antigen-binding fragment thereof can be in combination with a chemotherapeutic agent. Examples of chemotherapeutic agents include folinic acid (leucovorin), fluorouracil (5-FU), irinotecan, gemcitabine and nanoparticle albumin-bound paclitaxel (ABRAXANE). In some embodiments, the FOLFIRI combination: folinic acid, fluorouracil, and irinotecan, can be administered in combination with the humanized monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the initial dose of a chemotherapeutic agent in a method to treat a cancer in combination with a humanized monoclonal antibody or an antigen-binding fragment thereof can be administered prior to administration of an initial dose the humanized monoclonal antibody or an antigen-binding fragment thereof. In some embodiments, the initial dose of a chemotherapeutic agent in a method to treat a cancer in combination with a humanized monoclonal antibody or an antigen-binding fragment thereof can be administered after administration of an initial dose the humanized monoclonal antibody or an antigen-binding fragment thereof.

In some embodiments, an initial dose of the irinotecan can be about 180 mg/m² administered over about 90 minutes; an initial dose of the folinic acid is about 400 mg/m² administered over about 120 minutes and concurrently with the initial dose of the irinotecan; and/or an initial dose of the fluorouracil is about 400 mg/m² administered after administration of the initial dose of the folinic acid. In some embodiments, the folinic acid, fluorouracil, and irinotecan are administered every 14 days.

In some embodiments, the humanized monoclonal antibody or an antigen-binding fragment thereof can be administered in combination with an additional therapeutic agent. Examples of additional therapeutic agent include bevacizumab, aflibercept, cetuximab, and panitumumab.

EXAMPLES Example 1—Humanization of LGR5 Antibody

Human germline sequences were used as the acceptor frameworks for humanizing the murine antibody 18G7.1. To find the closest germline sequences, the most similar expressed light chain and the most similar heavy chain were identified in a database of germline sequences by NCI IgBLAST (ncbi.nlm.nih.gov/igblast/). In this search the CDR sequences of 18G7.1 were masked. The selection of the most suitable expressed sequence included checking for sequence identity of the canonical and interface residues, and checking for the similarity in CDR loop lengths.

In order to identify potential structural conflicts in key structural framework residues between the candidate humanized sequence and the parent murine monoclonal antibody 18G7.1, a three-dimensional model was generated. A composite of antibody structures was used to create a homology model with grafted candidate humanized sequences followed by molecular energy minimization. Structural analysis using computer software Pymol, was used to identify residues that could potentially negatively impact proper folding.

From this analysis, six candidate VH chains were constructed that included: 1) a functional human framework containing selected substitutions within the candidate humanized framework region based on analysis of likely impact on folding and ii) the parental 18G7.1 murine antibody CDRs (SEQ ID NOs: 1, 2, and 3). fused in-frame to the human IgG1 constant region are chemically synthesized.

Similarly, two candidate VL chains were constructed that included: 1) a functional human framework containing selected substitutions within the candidate humanized framework region based on analysis of likely impact on folding and ii) the parental 18G7.1 murine antibody CDRs (SEQ ID NOs: 4, 5, and 6). The candidate VL chain and the candidate VH chain fused in-frame to the human IgG1 constant region were chemically synthesized.

Selected candidate variant humanized heavy and light chain combinations were tested for functionality by co-transfection into mammalian cells. Each of the six candidate humanized 18G7.1 heavy chains described above were co-transfected with one of the candidate 18G7.1 light chains into HEK 293 cells, and conditioned media was assayed for LGR5 antigen binding activity by flow cytometry. In addition, three candidate humanized 18G7.1 heavy chains described above were co-transfected with the second candidate 18G7.1 light chain into HEK 293 cells, and conditioned media was assayed for LGR5 antigen binding activity by flow cytometry. The 18G7.1 candidate heavy chain/light chain combination (humanization variant) known as 18G7H6, and which exhibited the most robust binding was selected for affinity maturation.

Example 2—Humanized LGR5 Antibody Affinity Maturation

In order to increase the affinity of the selected humanized variant 18G7H6, a combination of alanine scanning mutagenesis and saturation mutagenesis was employed. Residues in heavy chain CDR1 and light chain CDR1 and CDR3 were mutated to alanine, transfected into HEK 293 cells, and the resultant conditioned media was assayed for LGR5 antigen binding activity by flow cytometry. Saturation mutagenesis was performed on heavy chain CDR3, in which every residue in CDR3 was mutated to each of the 19 naturally occurring amino acids except the original amino acid identity at that position. Each of the mutants were transfected into HEK 293 cells, and the resultant conditioned media was assayed for LGR5 antigen binding activity by flow cytometry.

These mutations were incorporated at increasing number into 3 constructs. These three constructs were then transfected into HEK 293 cells, and the resultant conditioned media was assayed for LGR5 antigen binding activity by flow cytometry. Two constructs 18G7H6A1 and 18G7H6A3 were selected for further characterization. TABLE 1A lists certain sequences of the antibodies.

TABLE 1A Description SEQ ID NO: 18G7.1 Heavy Chain CDR1 Amino Acid 1 18G7.1 Heavy Chain CDR2 Amino Acid 2 18G7.1 Heavy Chain CDR3 Amino Acid 3 18G7.1 Light Chain CDR1 Amino Acid 4 18G7.1 Light Chain CDR2 Amino Acid 5 18G7.1 Light Chain CDR3 Amino Acid 6 18G7H6A1 Heavy Chain DNA 7 18G7H6A1 Light Chain DNA 8 18G7H6A1 Heavy Chain Amino Acid 9 18G7H6A1 Light Chain Amino Acid 10 18G7H6A3 Heavy Chain DNA 11 18G7H6A3 Light Chain DNA 12 18G7H6A3 Heavy Chain Amino Acid 13 18G7H6A3 Light Chain Amino Acid 14 18G7Ch Heavy Chain DNA 15 18G7Ch Light Chain DNA 16 18G7Ch Heavy Chain Amino Acid 17 18G7ch Light Chain Amino Acid 18 18G7H6A3 Heavy Chain Variable Domain Amino Acid 19 18G7H6A3 Heavy Chain Variable Domain DNA 20 18G7H6A3 Light Chain Variable Domain 21 18G7H6A3 Light Chain Variable Domain DNA 22 18G7H6A3 Heavy Chain CDR1 Amino Acid 23 18G7H6A3 Heavy Chain CDR1 DNA 24 18G7H6A3 Heavy Chain CDR2 Amino Acid 25 18G7H6A3 Heavy Chain CDR2 DNA 26 18G7H6A3 Heavy Chain CDR3 Amino Acid 27 18G7H6A3 Heavy Chain CDR3 DNA 28 18G7H6A3 Light Chain CDR1 Amino Acid 29 18G7H6A3 Light Chain CDR1 DNA 30 18G7H6A3 Light Chain CDR2 Amino Acid 31 18G7H6A3 Light Chain CDR2 DNA 32 18G7H6A3 Light Chain CDR3 Amino Acid 33 18G7H6A3 Light Chain CDR3 DNA 34 18G7H6A1 Heavy Chain CDR1 Amino Acid 35 18G7H6A1 Heavy Chain CDR1 DNA 36 18G7H6A1 Heavy Chain CDR2 Amino Acid 37 18G7H6A1 Heavy Chain CDR2 DNA 38 18G7H6A1 Heavy Chain CDR3 Amino Acid 39 18G7H6A1 Heavy Chain CDR3 DNA 40 18G7H6A1 Light Chain CDR1 Amino Acid 41 18G7H6A1 Light Chain CDR1 DNA 42 18G7H6A1 Light Chain CDR2 Amino Acid 43 18G7H6A1 Light Chain CDR2 DNA 44 18G7H6A1 Light Chain CDR3 Amino Acid 45 18G7H6A1 Light Chain CDR3 DNA 46 LGR5 Amino Acid Sequence 47 18G7H6A1 Heavy Chain Variable Amino acid 48 18G7H6A1 Light Chain Variable Amino acid 49

Example 3—Production of Humanized LGR5 Antibodies

GS single gene vectors for 18G7H6A1, 18G7H6A3 and a chimeric 18G7.1 (murine Fab from 18G7.1 fused to human IgG1 Fc), named 18G7Ch were constructed, amplified and transiently co-transfected into Chinese Hamster Ovary cells (CHOK1SV GS-KO) using transient transfection for expression evaluation at a volume of 200 ml. Large scale transient transfection of CHOK1SV GS-KO cells at a final volume of 5 litres for 18G7CH and 2.5 litres for both 18G7H6A1 and 18G7H6A3 was then initiated. Clarified culture supernatant was purified using one-step Protein A chromatography. Product quality analysis in the form of SE-HPLC, SDS-PAGE and endotoxin measurement was carried out using purified material at a concentration of 1 mg/ml including an in-house human antibody as a control sample. Results showed high purity of product recovered (>95.7%).

Example 4—Construction of the Cell Line for a Humanized LGR5 Antibody

Stable GS-CHO transfectant pools, expressing the 18G7H6A3 antibody were created by transfection of CHOK1SV GS-KO host cells with the expression vector p18G7H6A3/DGV. The DGV containing the gene encoding the antibody was constructed, transfected and resultant clonal cell lines were subsequently generated by single cell sorting of the transfectant pools using a FACS method. The 96-well plates generated during cloning were screened weekly for the presence of single colonies. After approximately 2 weeks, supernatant from up 1000 colonies were screened for antibody production using an Octet® System method. Of the 1000 colonies screened, 991 produced detectable levels of antibody. The Octet data were ranked and the highest producing colonies were selected for progression.

The highest ranked colonies were progressed to suspension culture in 96-deep well plates in CD CHO medium and were subsequently adapted to subculture medium. Productivity of the selected cell lines were performed using a feed regime which mimicked, as closely as possible, the bioreactor process. The cultures were harvested on day 12 and assayed for antibody concentration using an Octet® System method. Antibody concentrations at harvest ranged from <20 mg/L to 3000 mg/L. Twenty cell lines were selected for further evaluation based upon rank position in the productivity screen, the parental pool from which the cell line was derived and evidence that each cell line arose from a single colony. The cultures of the 20 selected cell lines were expanded by serial subculture from 96 deep well plates to shake-flasks. Based upon rank position in the ‘abridged’ fed-batch suspension culture productivity screen and having acceptable growth characteristics during routine subculture in shake-flask cultures (consistently ≥1×106 viable cells per mL at routine subculture), the lead cell line selected for evaluation in two 10 L laboratory-scale stirred-tank bioreactors. This lead cell line demonstrated consistently high growth and viability during routine subculture and has >2000 mg/L titers at harvest. This cell line was used for creation of the Master Cell Bank (MCB) and for evaluation in 10 L laboratory-scale bioreactors

Example 5—Humanized LGR5 Antibody Binds to Human LGR5

A FACS-based assay was used to measure the binding of purified 18G7H6A1 and 18G7H6A3 to recombinant human LGR5 overexpressed on the surface of CHO cells. CHO and CHO-LGR5 cells were stained with serial dilutions of 18G7H6A1 or 18G7H6A3 at 4° C., surface staining was detected with PE-conjugated anti-human IgG secondary antibodies and analyzed on the FACScalibur. The EC50 of 18G7H6A1 and 18G7H6A3 for human LGR5 binding was <10 nM. An antibody control (MOPC) was used as a negative control in this experiment as well as wild-type CHO without LGR5. 18G7H6A3 showed no binding to the wild-type CHO and the isotype control did not show any measurable binding to human LGR5.

To identify potential animal model species for investigating the therapeutic efficacy and safety of 18G7H6A3, the cross-reactivity of 18G7H6A3 to LGR5 expressed by species homologues was determined in a series of in vitro binding studies. See FIG. 1. As shown, antibody 18G7H6A3 (BNC101) was found to strongly bind human and cyno LGR5, but not bind to rat or mouse LGR5.

Example 6—Binding of a Humanized LGR5 Antibody to Plate-Bound Recombinant, Human LGR5 Ectodomain

Binding of 18G7H6A1 and 18G7H6A3 to human LGR5 was assessed in vitro using an ELISA-based plate binding assay. The assay measured antibody binding to ELISA plate-bound purified recombinant, LGR5 ectodomain-IgG-Fc fusion, with detection of LGR5-bound antibody with horseradish peroxidase-conjugated anti-human IgG-CH1 secondary antibody. The EC50 of 18G7H6A3 for human LGR5-Fc was found to be 300 pM.

Example 7—Binding Characteristics of a Humanized LGR5 Antibody on Tumor Cells

The binding characteristics of 18G7H6A3 to human cancer cell lines expressing different levels of LGR5, were analyzed by flow cytometry to define the potential targeting properties of 18G7H6A3 on heterogeneous tumor populations. The expression levels of LGR5 in multiple tumor cell lines were quantified by flow cytometry.

Human tumor cell lines analyzed in these studies included colon carcinoma cancer cell lines (CT1 (Bionomics), CT3 (Bionomics), DLD1 (ATCC), Ls174T (ATCC), LoVo (ATCC), SW48 (ATCC), SW480 (ATCC), SW620 (ATCC) and HCT116 (ATCC)), triple negative breast cancer cell lines (Hs578T (ATCC) and MDA-MB-231 (ATCC)), pancreatic cancer cell lines (AsPC-1 (ATCC), BxPC3 (ATCC), Capan2 (ATCC), HPAFII (ATCC), SW1990 (ATCC), CFPAC (ATCC), Panc10.05 (ATCC) and PANC-1 (ATCC)), cisplatin-sensitive ovarian cancer cell lines (OVCAR3 (ATCC) and SK-OV-3 (ATCC)), cisplatin-resistant ovarian cancer cell lines (SK-OV-3/CP, OVCAR8/CP, Igrov1/CP and A2780/CP (TGEN)) and lung adenocarcinoma cell line HOP62 (ATCC).

Cells grown near confluence were lifted with TrypLE cell dissociation buffer (Life Technologies), counted and plated in 96-well V-bottom plates at 1×105 cells per well. 18G7H6A3 was tested at a starting concentration of 100 nM with serial dilutions in staining buffer (PBS/0.8% bovine serum albumin). Samples were incubated on ice for 30 minutes, then centrifuged at 1800 rpm for 2 minutes at 4° C. and washed 3 times with staining buffer. Fifty μk of secondary antibody goat anti-human IgG-PE conjugate at 1:250 dilution (Southern Biotech) was added to each corresponding well in staining buffer. Samples were incubated for an additional 15 minutes on ice, and then washed as described above and resuspended in 100 μl staining buffer containing propidium iodide (PI) (Life Technologies) for dead cell exclusion. Samples were analyzed on the FACScalibur flow cytometer using CellQuest (Becton Dickinson) and FlowJo (TreeStar, Inc) software.

The cell surface expression levels of LGR5 in multiple tumor cell lines were quantified by flow cytometry. CT1 colorectal tumor cells and pancreatic cancer cell lines Panc-1, Capan2 and CFPAC were among the highest LGR5 expressors. Moderate expression levels were observed in pancreatic cancer cell lines (AsPC-1, SW1990, HPAFII), cisplatin-resistant ovarian cancer cell lines (OVCAR8/CP, A2780/CP and Igrov1/CP) as well as colon, breast and ovarian cancer cell lines (SW48, Hs578T and OVCAR3). Low but detectable levels of LGR5 cell surface expression were observed in colon (SW480, LoVo) and breast cancer cell lines (MDA-MB-231). Table 2 summarizes the data for 18G7H6A3 FACS binding to Tumor cell lines.

TABLE 2 Tumor Cell line 18G7H6A3 (18G7.1) IgG CRC CT1 + − CT3 + − DLD1 +/− − Ls174T +/− − LoVo +/− − SW48 + − SW480 +/− − SW620 +/− − HCT116 +/− − Breast MDA-MB-231 +/− − MDA-MB-231 LM2 +/− − Hs578T + − CN34 +/− − CN34 LM1 +/− − Prostate PC-3 +/− − PCSD1 +/− − Ovarian OVCAR-3 + − SK-OV-3 +/− − SK-OV-3/CP +/− − OVCAR8/CP + − Igrov1/CP + − A2780/CP + − Lung HOP-62 +/− − Pancreatic AsPC-1 + − Capan2 ++ − HPAFII + − Swl990* + − CFPAC ++ − PANC-1 ++ −

Example 8—Inhibition of Cachectic Colorectal Tumor Growth In Vivo by a Humanized Anti-LGR5 Antibody

The CT1 primary CRC xenograft model was derived from a patient with stage IV metastatic colon cancer. DNA sequencing of this tumor identified common colon cancer mutations in multiple genes including K-Ras, PI3K, PTEN, p53 and APC. Low passage CT1 tumorspheres maintained in culture under serum-free conditions were injected into SCID/Bg mice in Matrigel subcutaneously on day 0, and monitored twice weekly for tumor size and body weight. At day 25 CT1 subcutaneous tumors were randomized into groups of 10 mice when tumors reached 120 mm3. Mice were treated with either PBS, antibody control MOPC, 18G7H6A1, 18G7H6A3 or human/murine chimeric 18G7Ch. Mice were dosed BIW at 15 mg/kg for 2.5 weeks (5 doses total).

Antibody 18G7H6A3 showed significant anti-tumor activity in vivo compared to PBS and MOPC antibody controls during the course of 4 doses (15 mg/kg, twice weekly). While antibody 18G7H6A1 showed anti-tumor activity, monoclonal 18G7H6A3 showed superior activity to both 18G7H6A1 and the parental murine chimeric 18G7Ch antibody. Table 3 shows percent CT1 tumor volume reduction (group vs MOPC) after 1-4 doses of Lgr5+ Abs.

TABLE 3 # of Doses: 1 2 3 4 18G7Ch 9.2% 30.6% 19.5% 29.0% 18G7H6A1 17.5% 19.1% 14.2% 19.0% 18G7H6A3 38.8% 42.0% 28.9% 35.4%

Example 9—Inhibition of Colorectal Tumor Growth In Vivo by a Humanized Anti-LGR5 Antibody

The CT3 primary CRC xenograft model was derived from a patient with stage III mCRC with mutations in K-Ras, H-Ras, APC, PI3K, PTEN, STK11, RB1, TP53, FGFR2, VANGL2, and ISCO. Low passage cryopreserved CT3 primary xenograft tumor fragments were implanted into 5 SCID/Bg mice. Tumors averaging ˜1150 mm3 pooled from five CT3 primary xenograft-bearing SCID mice were removed at day 41 post-implant, dissociated and re-implanted into CB.17 SCID mice in Matrigel subcutaneously, and monitored twice weekly for tumor size and body weight. When tumors reached an average of 130 mm3, mice were randomized (34 days post implant). Mice were treated with either PBS, antibody control MOPC, 18G7H6A3, 18G7H6A1 or human/murine chimeric 18G7Ch. Mice were dosed BIW at 15 mg/kg for 2.5 weeks (5 doses), starting on day 34. All mice were monitored twice weekly for body weight and tumor size, as well as overall health and appearance, until termination.

While antibody 18G7H6A1 showed anti-tumor activity, monoclonal 18G7H6A3 showed significant anti-tumor activity compared to PBS and MOPC antibody controls after 4 doses (15 mg/kg, twice weekly). 18G7H6A3 showed superior activity to the parental murine chimeric 18G7Ch antibody and equivalent activity to 18G7H6A1. Table 4 shows percent CT3 tumor volume reduction (group vs MOPC) after n dose of test Abs.

TABLE 4 # of Ab Doses: 1 2 3 4 18G7Ch 22.6% 8.9% 17.0% 13.8% 18G7H6A1 18.3% 12.6% 28.8% 28.7% 18G7H6A3 34.2% 38.1% 23.4% 28.2%

Example 10—Inhibition of Colorectal Tumor Growth In Vivo by a Humanized Anti-LGR5 Antibody in Combination with FOLFIRI

CB.17 SCID mice were implanted with CT3 cells grown under CSC conditions. At day 40 post-implantation, when tumors reached ˜160 mm3, mice were randomized into treatment groups including i) PBS, ii) FolFiri (5FU 30 mg/kg, leucovorin 90 mg/kg and Irinotecan 24 mg/kg), given every 5 days for 15 days (3 doses total), and iii) Combination of FolFiri (as in ii.) and 18G7H6A3 (15 mg/kg twice per week). Analyses of tumor volume showed that combination of 18G7H6A3 and FolFiri reduced growth of CT3 tumors compared to FolFiri regimen. Combination treatment reduced tumor volume at days 61, 65, 68, 71 and 75 by about 58%, 53%, 45%, 33% and 37% respectively (FIG. 2).

Example 11—Inhibition of Pancreatic Cancer Tumor Growth In Vivo by a Humanized Anti-LGR5 Antibody

To assess efficacy of 18G7H6A3 as single agent or in combination with standard of care, a pancreatic cancer xenograft model was tested. CB17.SCID mice were implanted with AsPC-1 cells (in matrigel+RPMI in a 1:1 ratio). Tumors were randomized at day 20 post implantation into 5 groups: i) PBS, ii) MOPC (15 mg/kg, twice per week, ip), iii) 18G7H6A3 (15 mg/kg, twice per week, ip), iv) gemcitabine (90 mg/kg, twice per week, ip) and v) concurrent combination of gemcitabine and 18G7H6A3 at the above doses.

It was discovered that 18G7H6A3 as single agent inhibited tumor growth compared to saline and/or control IgG up to nearly 40% at day 41 post implantation. In addition, the combination of 18G7H6A3 and gemcitabine significantly inhibited tumor growth in AsPC-1 model (up to 36% at day 61 post implantation) compared to gemcitabine alone. 18G7H6A3 as single agent also provided some inhibition in tumor growth compared to PBS and control IgG up to day 65.

Example 12—Inhibition of Triple Negative Breast Cancer Tumor Growth In Vivo by a Humanized Anti-LGR5 Antibody

This in vivo study was performed using low passage triple negative breast cancer cells (ER−, PR−, no HER2 overexpression). MDA-MB-231-LM3 cells were maintained in adherent culture with DMEM/10% FBS/anti-anti medium. CB.17 SCID mice were injected on day 0 with MDA-MB-231-LM3 cells in RPMI:Matrigel (1:1) into the 4th mammary fat pad and monitored twice weekly for tumor size and body weight. At day 27, MDA-MB-231-LM3 tumors were randomized into 4 groups of 10 mice when tumors reached ˜155 mm³. Mice were treated with PBS, antibody control MOPC, or 18G7H6A3. Mice were dosed BIW at 15 mg/kg for 3.5 weeks (7 doses). It was discovered that antibody 18G7H6A3 showed significant anti-tumor activity compared to PBS (60.7% tumor growth inhibition) or MOPC antibody (49.3% tumor growth inhibition) controls (FIG. 3).

Example 13—Induction of Expression of LGR5 in Colorectal Cancer Cells Treated with a SN38 or a PI3K/mTOR Inhibitor

A panel of CRC cell lines including DLD1, HCT116, LS174t, LoVo, SW48, SW480 and SW620 were treated with a PI3K/mTOR dual inhibitor (NVP) or 2 different cytotoxic agents including SN38 (active metabolite of Irinotecan) or 5FU (5 fluorouracil). Cells were treated with the above agents at 1 um and were harvested after 72 hrs. Cells were then stained with anti-LGR5 Mab conjugated to Alexa Fluor647 and the data were analyzed by flow cytometry using a FACScalibur.

Flow cytometry analyses of CRC cell lines showed greater expression of LGR5 in LoVo, HCT116, LS174t, SW48, SW480 and SW620 cells when treated with a PI3K/mTOR inhibitor. Additionally, treatment with SN38 promoted LGR5 expression in HCT116, LS174t, SW48, SW480 and especially SW620 cells. 5FU treatment, however, did not induce LGR5 expression in any of these lines suggesting that underlying mechanisms governing LGR5 expression are distinct in these lines. These data indicate that LGR5+ cells are more resistant to treatment with the above agents as treatments have mostly targeted the LGR5 negative non-cancer stem cell population. To understand if treatment with these agents upregulate LGR5 expression on these cells, we analyzed LGR5 cell surface expression by flow cytometry in all the cell lines. Upon treatment with PI3K/mTOR inhibitor, LGR5 expression was significantly upregulated in LoVo. These data indicate that treatment with small molecule inhibitors or cytotoxic agents target LGR5neg cells and causes increased expression of LGR5 in these cells.

Example 14—LGR5 Expression is Promoted in Pancreatic Cancer Cell Lines Treated with Small Molecule Inhibitors or Cytotoxic Agents

In addition to CRC cell lines to further expand the above findings, expression of LGR5 was investigated in a series of pancreatic cell lines treated with relevant standard of care including nab-paclitaxel, gemcitabine and taxol and also small molecule inhibitors targeting most relevant pathways in pancreatic cancer such as inhibitors of PI3K, MEK and GSK3β. The pancreatic cell lines that were tested include: AsPc1, HPAFII, PANC1, BxPC3, CFPAC, PANC10.05, Capan2 and SW1990. Treatment with nab-paclitaxel results in LGR5 upregulation in PANC1, BxPc3 and PANC10.05 as assessed by flow cytometry. Gemcitabine treatment upregulates LGR5 in PANC1 and taxol treatment results in increased LGR5 expression in HPAFII. The PI3K/mTOR treatment results in upregulation of LGR5 in CFPAC and the MEK inhibitor upregulates LGR5 in HPAFII and SW1990.

Example 15—LGR5 is Upregulated in Colorectal Cancer Tumors Treated with FOLFIRI Regimen (5FU, Leucovorin and Irinotecan)

To investigate if chemo treatment alters LGR5 expression in colorectal tumors, mice were treated every 5 days with 5FU (30 mg/kg i.p), leucovorin (90 mg/kg) and 2 different doses of irinotecan (24 mg/kg or 8 mg/kg). The result of those studies showed that while CT3 tumors were sensitive to the chemo regimen, CT1 tumors did not full regress and showed some resistance to the regimen (FIG. 4). To examine the effect of FOLFIRI treatment of LGR5 expression, total mRNA was extracted from CT1 and CT3 patient derived tumors and expression of LGR5 and was determined by qRT-PCR and was analyzed by subtracting the Ct value (cycle threshold) of LGR5 in each sample from its corresponding GAPDH transcript to generate DCT (delta Ct) values. Data are presented as 2 to the power of DCT. Analyses of abundance of LGR5 showed that the LGR5 transcript is increased in both CT1 (for about 2 folds) and CT3 tumors (approximately 3.5 folds) compared to corresponding saline treated tumors.

Example 16—LGR5 is Upregulated in Pancreatic Cancer Tumors Treated with Gemcitabine Alone and in Combination of Nab-Paclitaxel

To investigate if standard of care chemotherapy treatment for pancreatic cancer alters LGR5 expression in pancreatic tumors, mice were treated twice per week with combination of gemcitabine and nab-paclitaxel (in JH109 primary xenografts). At terminal analysis, qRT-PCR data using tumor cDNA showed a remarkable increase in the expression of LGR5 in chemotherapy treated tumors compared to corresponding saline-treated tumors indicating that treatment with standard of care results in upregulation of LGR5 in tumor cells.

LGR5 expression in JH109 model which is a patient derived xenograft model of pancreatic tumor. Mice were implanted with tumor chunks that were continuously passaged in the recipient but were never exposed to in vitro culture condition. Treatment of tumor-bearing mice with a chemotherapy regimen (combination of gemcitabine and nab-paclitaxel) resulted in a significant inhibition in tumor growth. Consistent with the colon cancer models, chemotherapy resulted in upregulation of LGR5 (more than 4-fold) in JH109 tumors, further suggesting enrichment of the cancer stem cell population upon treatment with chemotherapy. See, for example, FIG. 5.

Example 17—Inhibition of Pancreatic Tumor Growth In Vivo by a Humanized Anti-LGR5 Antibody

Efficacy of 18G7H6A3 was also investigated in a pancreatic cancer xenograft model. CB.17 SCID mice were implanted with PANC1 cells (1E6/mouse s.c in matrigel+RPMI 1:1 ratio), and randomized at day 41 post implantation into treatment groups: i) PBS, ii) IgG control (15 mg/kg, twice per week, ip), iii) 18G7H6A3 (15 mg/kg, twice per week, ip), iv) gemcitabine (90 mg/kg, twice per week, ip) and v) concurrent combination of gemcitabine and 18G7H6A3 (15 mg/kg, twice per week, ip). Gemcitabine was administered in assigned group for 3 weeks to inhibit tumor growth. All mice were monitored twice weekly for body weight and tumor size, as well as overall health and appearance.

Analysis of tumor volume showed that while there is a trend in favor of 18G7H6A3 as single agent (up to 30% at day 70 post implantation) to inhibit tumor growth, combination of 18G7H6A3 and gemcitabine significantly inhibited growth of PANC1 tumors (up to 52% at day 80 post implantation) compared to gemcitabine alone group. See FIG. 6.

In this example, the significant activity of 18G7H6A3 observed when administered in combination with chemotherapy (gemcitabine) can be attributed to the increased expressed of the target antigen LGR5 in response to gemcitabine treatment.

Example 18—Inhibition of Pre-Treated Pancreatic Tumor Growth In Vivo by a Humanized Anti-LGR5 Antibody

In addition to cell lines, we also investigated the efficacy of 18G7H6A3 as single agent or in combination with standard of care in the JH109 primary patient derived xenograft model of pancreatic cancer. The JH109 xenograft model is from a patient that had received four treatment regimens including 5-FU, Gemcitabine, Erbitux and radiotherapy. The original patient tumor has been passaged in immune-deficient mice continuously without any exposure to in vitro culture. To test efficacy of 18G7H6A3 in JH109 model, tumor bearing mice (n=7) were treated with control IgG (15 mg/kg i.p twice/week), 18G7H6A3 (15 mg/kg i.p twice/week) single agent, standard of care chemo (combination of gemcitabine (50 mg/kg i.p once week; and nab-paclitaxel 30 mg/kg, i.v once a week), combination of chemo and control IgG, and combination of chemo and 18G7H6A3. While single 18G7H6A3 mAb did not affect tumor growth, combination of 18G7H6A3 with Nab-paclitaxel and gemcitabine chemotherapy led to a significantly greater degree of tumor inhibition compared to chemotherapy alone. 18G7H6A3 combined with chemotherapy led to 77% greater tumor growth inhibition compared to chemotherapy alone. Three mice treated with the 18G7H7A3 chemotherapy combination had complete eradication of their tumor (no measureable tumor detected). The 18G7H6A3 chemotherapy combination group continued to suppress tumor growth even after discontinuation of treatment and one mouse was still devoid of any measurable tumors three months after cessation of chemotherapy. In this example, the significant activity of 18G7H6A3 observed when administered in combination with chemotherapy (gemcitabine plus nab-paclitaxel) can be attributed to the increased expressed of the target antigen LGR5 in response to gemcitabine nab-paclitaxel treatment and is a demonstration of prevention of re-growth or recurrence of a primary tumor in vivo after chemotherapy treatment to eradicate the primary tumor bulk.

Example 19—Humanized LGR5 Antibody Treatment Reduces Cancer Stem Cell Populations

For flow cytometric analysis, cells from 5 individual tumors were stained with a variety of antibodies against stem cell specific markers CD44, and CD166. Tumors were dissociated, depleted for mouse cells and then counted for viable cells. Dissociated cells were used for analysis of cell surface stem cell marker expression by flow cytometry.

There was a decrease in cancer stem cell population as defined by CD166+/CD44+, LGR5+/CD166+, or LGR5+/CD166+/CD44+ subpopulations (FIG. 7).

Example 20—Humanized LGR5 Antibody Treatment Reduces Colon Cancer Tumor Recurrence and Cancer Stem Cell Frequency In Vivo

The effects of 18G7H6A3 in combination with FolFiri were tested in colon cancer CT3 model (Example 10). The results of this primary tumor efficacy study showed that 18G7H6A3 in combination with a 3 cycle FOLFIRI regiment was more effective than FolFiri alone in reducing tumor growth. To determine if the 18G7H6A3 FOLFIRI combination regimen was also effective in reducing cancer stem cell (CSC) frequency, tumors from day 78 were harvested, dissociated, pooled and re-implanted in a limiting dilution assay at 10, 30, 100 cells/flank into a new cohort of tumor naïve CB17.Scid mice. The mice were then monitored 2× per week for tumor growth, and tumors allowed to grow with no further treatment.

Cells isolated from mice treated with anti-LGR5 antibody 18G7H6A3 in combination with FOLFIRI had greatly decreased tumorigenicity as compared to cells isolated from mice treated with FOLFIRI alone (FIG. 8). In addition, the re-implanted cells from the 18G7H6A3 FOLFIRI combination had a significantly slower tumor growth profile and a delayed time to progression (FIG. 9) compared to FOLFIRI alone. Finally, the 18G7H6A3 treatment reduced cancer stem cell frequency by a linear regression analysis by a factor of 6 at day 40 (1/856.3 18G7H6A3/FOLFIRI vs 1/138.6 for FOLFIRI). These data indicate that 18G7H6A3 in combination with FOLFIRI effectively targets the tumor initiating or cancer stem cell population. Day 68 was the last day for the 30 cells/animal data. The data are significant at p=0.0039.

Example 21—Humanized LGR5 Antibody Treatment Reduces Pancreatic Cancer Tumor Recurrence and Cancer Stem Cell Frequency In Vivo

The effects of 18G7H6A3 in combination with gemcitabine were tested in pancreatic cancer PANC1 model. This study showed that 18G7H6A3 in combination with gemcitabine significantly inhibited tumor growth in PANC1 model compared to gemcitabine alone. Tumors cells from these treatment groups were harvested, dissociated, pooled and re-implanted in a limiting dilution assay (500, 1500, 4500 or 13500 cells/animal) into a new cohort of CB.17 SCID mice and allowed to grow with no treatment.

Cells isolated from mice treated with anti-LGR5 antibody 18G7H6A3 in combination with gemcitabine had greatly decreased tumorigenicity in the limiting dilution assay re-implant as compared to cells isolated from mice treated with gemcitabine alone. Re-implanted PANC1 tumors treated with combination of gemcitabine and 18G7H6A3 showed reduction in the frequency of engraftment in mice implanted with 4500 cells (40% in gemcitabine vs. 20% in combination) and also in mice implanted with 13500 cells (100% in gemcitabine vs. 70% in combination). Using linear regression, frequency of cancer stem cell in gemcitabine implanted tumors was about 1.5 fold higher in gemcitabine compared to combination group (1 in 14883 vs. 1 in 21336). These data indicate that 18G7H6A3 in combination with gemcitabine effectively targets the tumor initiating or cancer stem cell population.

In addition to PANC1 tumors, we also analyzed percentage of engraftment and cancer stem cell frequency in an limiting dilution experiment (using 500, 1500, 4500 and 13500 cells) in mice bearing AsPC-1 tumors treated with gemcitabine as single agent or in combination with 18G7H6A3. Tumor volume measurement at day 40 post treatment showed a reduction in percentage of tumor bearing mice in gemcitabine vs. combination in mice implanted with 4500 or 13500 cells (40% and 80% vs. 30% and 50%, respectively). Frequency of cancer stem cells was also greater by more than 1.5 fold in gemcitabine vs. combination group further indicating that 18G7H6A3 in combination with gemcitabine is targeting cancer stem cell population in pancreatic cancer.

Example 22—Humanized LGR5 Antibody Treatment Reduces Triple Negative Breast Cancer Tumor Recurrence and Cancer Stem Cell Frequency In Vivo

The effects of 18G7H6A3 in combination with paclitaxel were tested in the triple negative breast cancer MDA-MB-231-LM3 model (Example 12). This study showed that 18G7H6A3 in combination with paclitaxel had minimal additive inhibition in tumor growth compared to paclitaxel alone. These tumors were harvested, dissociated, pooled and re-implanted in a limiting dilution assay at 10, 30, 100 cells/flank into a new cohort of CB.17 SCID mice and allowed to grow with no treatment.

Cells isolated from mice treated with anti-LGR5 antibody 18G7H6A3 in combination with paclitaxel had greatly decreased tumorigenicity as compared to cells isolated from mice treated with paclitaxel alone. In addition, the re-implanted cells from the 18G7H6A3 plus paclitaxel tumors had a significantly slower tumor growth profile and a delayed time to progress compared to paclitaxel alone. Finally, the 18G7H6A3 plus paclitaxel treatment reduced cancer stem cell frequency by a linear regression analysis. These data indicate that 18G7H6A3 in combination with paclitaxel effectively targets the tumor initiating or cancer stem cell population.

Example 23—Inhibition of Metastatic Colorectal Cancer Growth In Vivo by Prophylactic Treatment with Humanized Anti-LGR5 Antibody and Chemotherapy

The in vivo study was performed using low passage colorectal cancer cells (BMCRC086) derived from a liver met of a patient with colorectal cancer. On Day 0, BMCRC086 cells were thawed, suspended in RPMI:Matrigel (1:1) and injected subcutaneously into the rear flank of CB.17 SCID mice. Animals were monitored twice weekly for tumor size and body weight. At day 7, mice were treated with PBS, 18G7H6A3, FOLFIRI or FOLFIRI in combination with 18G7H6A3. Mice were dosed with PBS and 18G7H6A3, BIW at 15 mg/kg for 7.5 weeks (16 doses). Mice were dosed with FOLFIRI (30 mg/kg Fluorouracil and 90 mg/kg Leucovorin on days 7, 12, 17, 22, 27 and 32; 24 mg/kg Irinotecan on days 8, 13, 18, 23, 28 and 33) for 4 weeks (6 doses). 18G7H6A3 in combination with FOLFIRI showed significant anti-tumor activity compared to FOLFIRI alone (FIG. 10).

Example 24—Humanized LGR5 Antibody Treatment Inhibits Wnt Signaling Pathways

18G7H6A3 treated tumors from colon cancer CT1 (Example 8) and CT3 (Example 9) in vivo tumor efficacy studies were characterized by western blot analysis. Tumor samples from each treated mouse (n=5 to 10 mice per group) were resected after sacrificing, immediately frozen in a liquid nitrogen cooled mortar, ground-up pestle (cryopulverization), flash frozen in liquid nitrogen and stored at −80° C. until used. Cryopulverized tumors were lysed with ice cold lysis buffer (reducing RIPA buffer containing phosphatase and protease inhibitors) for 30 minutes on ice with occasional vortexing. Supernatants containing tumor lysate protein were run on a SDS-PAGE gel followed by western blotting for a number of Wnt-signal proteins (and their phosphorylated forms). A number of significant differences between treatment groups were observed in western blots of CT1 and CT3 tumors. In FIG. 11, phospho-Thr41/Ser45-β-catenin (a Wnt-signal protein) is a marker of inactive, and subsequently degraded, form of the protein demonstrating 18G7H6A3 is able to inhibit LGR5 signaling in tumor cells in vivo.

Example 25—Humanized LGR5 Antibody Treatment Does Not Inhibit In Vitro Wnt-Signaling Pathway

Parental HEK-293T cells and HEK-293T cell stably expressing LGR5 were transduced with a TCF-LEF reporter vector-containing lentivirus (GFP Cignal, QIAGEN) and selected for stable expression of the reporter. Parental and LGR5 expressing stable reporter lines were plated at 25,000/well in a 96 well plate, attached overnight, serum starved and treated with antibodies or vehicle for 6 h, then treated with recombinant human Wnt3a (3 nM) and recombinant human R-spondins for 18 h. Two concentrations for each R-spondins1-3 and one concentration of R-spo4 were tested (100 pM, 300 pM, 1 nM, 3 nM or 10 nM) based on our analysis of the activity of the different R-spondins in activation of the TCF/LEF reporter cell lines. The reporter driven GFP signal was measured on a plate reader. All experiments shown are pooled data from three independent experiments (each experiment performed in duplicate) for each R-spondin tested (data are means+SD).

As shown in FIG. 12, increasing concentrations of soluble 18G7H6A3 did not affect the induction of TCF/LEF promoter driven GFP expression by the combination of Wnt3a plus RSPO1, RSPO2 or RSPO3. A positive control antibody 76C12, which has been shown to inhibit the induction of signaling activity through both LGR4 and LGR5 in the presence of RSPO and Wnt, is also shown. This data demonstrates that the anti-LGR5 antibody 18G7H6A3 does not block RSPO-driven TCF/LEF promoter activation.

Example 26—Humanized LGR5 Antibody Targets Tumor Cells Via ADCC (Antibody Dependent Cell Cytotoxicity) Mechanism

CHO-LGR5 cells were grown to confluent and were spun down, resuspended in PBS and were counted. An aliquot of cells (approximately 100 k) were added to another tube containing 100 μM pre-warmed (37° C.) CFSE (Carboxyfluorescein succinimidyl ester) and the mixture was incubated in the cell incubator for 15 min. The final CFSE concentration was about 1 μM. Next, cells were washed and resuspended in pre-warmed medium and were placed in the incubator for another 30 minutes followed by washing with PBS. The stained cells were then stained with 18G7H6A3 (100 μM). To ensure binding of the antibody to CHO-LGR5 cells, in some studies an aliquot of cells was also stained with a secondary goat anti-human PE conjugated antibody and was analyzed on the calibur machine in the laboratory. The U937 cells were stained with DDAO-SE (DDAO succinimidyl ester; 2 μM of dye for 100K cells) for 15 minutes and in a light protected place in the laboratory and at room temperature. Cells were then 1 ml of FBS (fetal bovine serum) followed by incubation in a light protected place for 5 minutes. Next, cells were washed with PBS supplemented with FBS (10%) and were resuspended in RPMI supplemented with FBS (2.5%). Both CHO-LGR5-18G7H6A3 and U937-DDAO-SE labeled cells were co-incubated in the cell incubator for 5 hrs and were analyzed in the calibur machine in the laboratory. As a negative control, an aliquot of CHO-LGR5-CFSE cells (no 18G7H6A3 staining) was also co-incubated with U937 and was analyzed on the calibur machine.

Analysis of flow cytometry data showed that majority of CHO-LGR5 cells stained with CFSE and 18G7H6A3 are viable and detectable in the calibur machine. Additionally, both U937 (U937 (human monocyte cell line; effector cells) and CHO-LGR5 cells were detectable when stained and were acquired individually. Finally co-incubation of U937-DDAO-SE and CHO-LGR5-CFSE-18G7H6A3 identified a double positive population of cells, however, co-incubation of U937 and CHO-LGR5-CFSE which lacks 18G7H6A3 did not generate the double positive population. The presence of the double positive population is indicative of a cross binding of U937 (which express FcR) to CHO-LGR5-18G7H6A3 (which express Fc portion) and further suggests that ADCC is one of the mechanisms of anti-tumor activity of 18G7H6A3.

Example 27—Humanized LGR5 Antibody Internalizes LGR5

Internalization of 18G7H6A3 was examined on CHO cell overexpressing LGR5. Cells were stained with 100 nM antibody for 30 min-2 hrs at 4° C., excess Ab was washed off and stained cells were incubated at either 4° C. or 37° C. Cells were stained with AlexaFluor488-conjugated secondary antibodies at various time points to monitor internalization of cell surface-bound antibodies. Upon incubation at 37° C., the internalized rate had a measured t½ value for surface localization of 6.703±1.282 minutes. Internalization was largely blocked by incubation at 4° C. although some decrease in surface-bound antibody was observed.

Example 28—Humanized LGR5 Antibody Does Not Competitively Block Binding of Soluble RSPOs to LGR5

Interaction of biotin-18G7H6A3 with hLGR5-Fc in the presence of human R-spondin 1/2/3/4 proteins was examined using competition ELISA format. LGR5-Fc was coated on a 96-well high binding ELISA plate at 2 μg/mL, and incubated overnight at 4 C. The plate was blocked with PBS+1% BSA. Biotin-18G7H6A3 was diluted in binding buffer to 1 μg/mL. The concentration was chosen from previous direct binding ELISA between LGR5-Fc and biotin-18G7H6A3 to give robust signal above EC50 concentration. Competitor proteins were added to the ELISA plate at the same time as biotin-18G7H6A3 at varying concentrations. A dilution of 1:1,000 of streptavidin-HRP (R&D Systems, cat #890803) was used for detection. Plate was developed with TMB (Thermo), and data were collected on SpectraMax Plus 384 plate reader at 450 nm. Data analysis was done using GraphPad Prism 6 program. The ELISA was repeated three times with some modifications of biotin-mAb and competitor concentrations.

As a positive control, LGR5-Fc was competed with the binding of biotin-18G7H6A3 to hLGR5-Fc on the plate. R-spondins 1/2/3/4 were tested for the ability to block binding of biotin-18G7H6A3 to LGR5-Fc coated on the plate. The proteins were purchased from R&D Systems, and are full length constructs expressed in mammalian cells. At the highest concentration of R-spondin proteins, complete blocking of antibody binding to LGR5 was not observed (FIG. 13).

Example 29—Humanized LGR5 Antibody Does Not Competitively Block Binding of Soluble RSPOs to LGR5

Binding of ligand alone (RSPO or Norrin) to LGR5 is not sufficient to induce LGR5 signaling. Instead, LGR5 forms ternary complexes with multiple co-receptors to drive signaling. To examine the effects of 18G7H6A3 on the formation of LGR5 ternary complexes, the binding of LGR5 to RNF43, ZNRF3, and LRP6 in the presence of R-spondin 1/2/3/4 and Norrin was examined using an ELISA format. RNF43-Fc, ZNRF3-Fc, and LRP6-Fc were coated on a 96-well high binding plate at 4 μg/mL in 1×PBS. The plate was incubated overnight at 4° C. and blocked with PBS+1% BSA. LGR5-Fc was diluted in primary buffer to 1 μg/mL, all in the presence or absence of 1 μg/mL of R-spondin 1/2/3/4 or 0.5 μg/mL of Norrin. R-spondin 1/2/3/4 or Norrin were preincubated together with hLGR5-Fc before being added to the ELISA plate. Triplicate wells were used for each condition was tested in triplicate. 1:2,000 anti-FLAG mAb (Cell Signaling) was used to detect bound hLGR5-Fc. 1:10,000 dilution of anti-mouse IgG HRP (JIR) was used for detection. Plate was developed with TMB (Thermo), and data were collected on SpectraMax Plus 384 plate reader at 450 nm. Data analysis was done using GraphPad Prism 6 program. Formation of a ternary complex with LGR5, ligands RSPO or Norrin, and co-receptor (RNF43-Fc, ZNRF3-Fc, and LRP6-Fc) was observed.

Next, 18G7H6A3 was added in addition to the ELISA plate in the presence of LGR5-Fc and RSPO or Norrin. 18G7H6A3 significantly reduced the formation of LGR5 ternary complexes with both RSPO and Norrin ligands as well as all three co-receptors (RNF43, ZNRF3, and LRP6). See FIG. 14. As 18G7H6A3 does not directly or competitively compete with ligand binding, this data is evidence of an allosteric model of inhibition.

Example 30—Epitope Mapping of Anti-LGR5 Antibody 18G7H6A3

To further characterize the specific region(s) of LGR5 that antibody 18G7H6A3 binds, an epitope mapping experiment was performed using hydrogen deuterium exchange mass spectrometry. Prior to conducting the hydrogen-deuterium exchange experiments, test digests prepared with undeuterated buffer in varying concentrations of guanidine hydrochloride (GdnHCl) were made to optimize proteolysis conditions for the best peptide coverage of LGR5 alone. For pepsin digestion for DXMS, a sample was thawed at 5° C. and then immediately digested over a protease column filled with porcine pepsin (Sigma) at a flow rate of 100 μl/min with 0.05% trifluoroacetic acid. Peptic fragments were collected on a C18 trap column and separated on a C18 reversed phase column (Vydac) with a linear acetonitrile gradient from 6 to 38%. The column effluent was electrosprayed directly into an LCQ Classic (Thermo Finnigan, Inc.) or Q-TOF mass spectrometer (Micromass). Determination of pepsin-generated peptides from MS/MS data sets was facilitated through the use of SEQUEST (Thermo Finnigan, Inc.). This set of peptides was then further verified by DXMS Explorer (Sierra Analytics Inc., Modesto, Calif.). The peptide coverage maps for the different concentrations of GdnHCl were compared, and the condition with the best coverage map for each individual protein or protein complex was used for subsequent deuterium exchange experiments. All steps were performed at 0° C. as described previously.

Exchange experiments were initiated by mixing LGR5-Fc in protein buffer, or LGR5-Fc preincubated with 18G7H6A3 with D20 buffer to a final concentration of 50% D20. The mixtures were incubated at 0° C. for 10, 30, 100, 300, 1,000, 3,000, or 10,000 s and then the exchange reaction was quenched by adding ice-cold quench solution (0.96% formic acid, 0˜0.8 M guanidine hydrochloride) resulting in samples with final concentrations of 0.58% formic acid and 0˜0.5 M guanidine hydrochloride, pH 2.5. The samples were then immediately frozen on dry ice and stored at −80° C. Data processing of DXMS experiments utilized specialized software as previously described (DXMS Explorer, Sierra Analytics Inc.).

The hydrogen/deuterium (H/D)-exchange data provide details regarding changes in solvent exposure due to binding of 18G7H6A3 and the burying of surface exposed residues upon binding of antibody to antigen. The HD exchange data analysis indicates that 18G7H6A3 binds to amino acids T175, E176, Q180, R183, 5186, A187, Q189, D247, E248, T251, R254, 5257, N258, K260 of SEQ ID NO: 47 within the convex surface of leucine rich repeats 6-9, on the opposite of the face of the R-spondin binding site as identified by X-ray crystallographic studies. (See e.g., Chen et al. Genes Dev. 27(12):1345-50 which is incorporated by reference in its entirety). These data show that the residues involved in binding of LGR5 to the R-spondins are not involved in binding 18G7H6A3. These preliminary results do not preclude that fact that other structural elements in LGR5 may be involved in the binding site of 18G7H6A3.

Example 31—Administration of 18G7H6A3 to a Human Patient Suffering from Colon Cancer

A population of human patients suffering from colon cancer is treated with chemotherapy and tumor volume is monitored. It is observed that average tumor volume ceases to expand and in fact decreases upon initiation of chemotherapy. Following an extended duration of time, the tumor volume stabilizes and eventually begins to increase.

A second human patient population suffering from colon cancer is treated with chemotherapy co-administered with 18G7H6A3. Again, average tumor volume is monitored. It is observed that tumor volume ceases to expand and in fact decreases upon initiation of chemotherapy. It is observed that tumor volume decreases to a minimum volume that is substantially lower than that of the first population. It is also found that tumor size remains low for a substantially extended period of time relative to the first population.

Example 32—Administration of 18G7H6A3 to a Human Patient Suffering from Colon Cancer

A first population of human patients suffering from colon cancer is administered chemotherapy alone. A second population of human patients suffering from colon cancer is administered chemotherapy in combination with 18G7H6A3.

The first population demonstrates a temporary reduction in tumor size and growth, after which tumor growth resumes and symptoms return. Tumor growth after chemotherapy treatment is recalcitrant to subsequent chemotherapy treatments.

The second population demonstrates reduction in tumor size to a basal level and cessation of tumor growth. Tumor growth does not resume during or upon completion of a treatment regimen. After completion of the regimen, growth does not return and symptoms of the cancer are no longer present in the second population.

Example 33—Administration of 18G7H6A3 to a Human Patient Suffering from Colon Cancer Increases Survival

A first population of human patients suffering from colon cancer is administered chemotherapy alone. A second population of human patients suffering from colon cancer is administered chemotherapy in combination with 18G7H6A3.

Patient survival at a set duration after treatment (1 year) is monitored. It is observed that patient survival in the second population is substantially higher than patient survival in the first population. That is, a significantly higher proportion of the second population survives past the first year after treatment as compared to the survival rate of the first population.

Similar observations are made at later intervals, and it is observed that among survivors at the first interval, members of the second group are significantly more likely to survive to a second interval (2 years after treatment) that are members of the first group alive at 1 year post treatment.

Example 34—Administration of 18G7H6A3 to a Human Patient Suffering from Breast Cancer

A population of human patients suffering from breast cancer is treated with chemotherapy and tumor volume is monitored. It is observed that average tumor volume ceases to expand and in fact decreases upon initiation of chemotherapy. Following an extended duration of time, the tumor volume stabilizes and eventually begins to increase.

A second human patient population suffering from breast cancer is treated with chemotherapy co-administered with 18G7H6A3. Again, average tumor volume is monitored. It is observed that tumor volume ceases to expand and in fact decreases upon initiation of chemotherapy. It is observed that tumor volume decreases to a minimum volume that is substantially lower than that of the first population. It is also found that tumor size remains low for a substantially extended period of time relative to the first population.

Example 35—Administration of 18G7H6A3 to a Human Patient Suffering from Breast Cancer

A first population of human patients suffering from breast cancer is administered chemotherapy alone. A second population of human patients suffering from breast cancer is administered chemotherapy in combination with 18G7H6A3.

The first population demonstrates a temporary reduction in tumor size and growth, after which tumor growth resumes and symptoms return. Tumor growth after chemotherapy treatment is recalcitrant to subsequent chemotherapy treatments.

The second population demonstrates reduction in tumor size to a basal level and cessation of tumor growth. Tumor growth does not resume during or upon completion of a treatment regimen. After completion of the regimen, growth does not return and symptoms of the cancer are no longer present in the second population.

Example 36—Administration of 18G7H6A3 to a Human Patient Suffering from Breast Cancer Increases Survival

A first population of human patients suffering from breast cancer is administered chemotherapy alone. A second population of human patients suffering from breast cancer is administered chemotherapy in combination with 18G7H6A3.

Patient survival at a set duration after treatment (1 year) is monitored. It is observed that patient survival in the second population is substantially higher than patient survival in the first population. That is, a significantly higher proportion of the second population survives past the first year after treatment as compared to the survival rate of the first population.

Similar observations are made at later intervals, and it is observed that among survivors at the first interval, members of the second group are significantly more likely to survive to a second interval (2 years after treatment) that are members of the first group alive at 1 year pot treatment.

Example 37—Administration of 18G7H6A3 to a Human Patient Suffering from Colon Cancer Decreases Side Effects

A first population of human patients suffering from colon cancer is administered chemotherapy and an anti-LGR5 antibody that blocks LGR5-RSPO binding and signaling. A second population of human patients suffering from colon cancer is administered chemotherapy and 18G7H6A3.

The first population demonstrates non-therapeutic side effects associated with the interference of RSPO1 signaling through LGR5. These side-effects are detrimental to patient health.

The second population, administered 18G7H6A3 in combination with chemotherapy, does not demonstrate non-therapeutic side effects associated with the interference of RSPO1 signaling through LGR5.

Example 38—LGR5 Expression in Advanced CRC Tumors

LGR5 transcript expression was investigated using RNAscope technology with LGR5 specific probes. LGR5 transcript was detectable in tissues including colon, intestine, cerebellum and pancreas. LGR5 transcript was also detectable in patient derived xenograft (PDX) tissues including CT1 CRC and JH109 pancreatic tumors. LGR5 expression was investigated in CRC patient samples isolated at different stages of tumorigenesis including early (Grade-I) vs. advanced (Metastatic) lesions. LGR5 transcript was expressed in CRC Grade I, II and II lesions, and was highly expressed in CRC metastatic lesions.

Example 39—LGR5 Expression in Metastatic Pancreatic Patient Derived Xenografts

LGR5 expression in metastatic pancreatic patient derived xenografts was investigated using the quantitative polymerase chain reaction (QPCR). A sample of tumor tissue was flash frozen or added to a cryovial containing RNAlater (Qiagen, CA), and transferred to −70° C. after incubation at 4° C. for several hours. Total RNA was extracted using a Qiagen RNeasy extraction kit (Qiagen, CA), and cDNA was synthesized using a SuperScriptIII kit (Life Technologies, CA) and protocols provided by the manufacturer. Human LGR5 transcript abundance was measured using human specific LGR5 and GAPDH primers and the following thermal condition in the StepOne Thermocycler (Life Technologies, CA): 50° C. (2 min); 90° C. (2 min) and 40 cycles of 90° C. (15 sec) and 60° C. (1 min) and melt curve assessment (from 65° C.-95° C.). LGR5 abundance was quantified using 2̂δCt equation.

LGR5 was highly expressed in metastatic pancreatic patient derived xenografts. Treatment with chemotherapy resulted in increased LGR5 expression in pancreatic tumors. Using human specific primers, LGR5 transcript was measurable using QPCR in a series of pancreatic patient derived xenografts. While LGR5 was detectable in most tumors there was a trend for increased LGR5 expression in metastatic tumors further suggesting a role for LGR5 in advanced tumorigenesis.

LGR5 expression was investigated in a series of pancreatic tumors including JH109, ASPC1 and PANC1. Treatment with a standard of care treatment (SOC) (Gemzar and Abraxane in JH109 and Gemzar alone in PANC1 and ASPC1) resulted in an induction in LGR5 expression in each of the foregoing tumors (FIG. 15). Notably, LGR5 expression was reduced to levels comparable to controls (saline or MOPC) in tumors treated with combination of 18G7H6A3 and SOC. These data further indicate that LGR5 expression can serve as a biomarker of response to combination therapy (18G7H6A3+SOC) in PANC tumors.

Example 40—CTNNB1 is One of the 18G7H6A3 Target Genes in CRC and Pancreatic Tumors

Potential targets in the Wnt pathway for 18G7H6A3 were investigated. Wnt QPCR plates (Qiagen, CA) were prepared with primers for about 80 Wnt pathway genes in a 96 well PCR plate. cDNA from 18G7H6A3 or MOPC (control) treated tumors was pooled and QPCR in the Wnt plate was performed. Data in each plate was normalized to corresponding GAPDH and the abundance of each gene was measured using an 2̂δCt equation. To measure fold differences, data in each 18G7H6A3 treated tumor was divided by the corresponding value from MOPC treated group. Values above 1 or below 1 were indicative of upregulation or downregulation in 18G7H6A3 treated group, respectively. Preliminary assessment of the number of genes that were up- or down-regulated showed that in both tumor models (CT1 and CT3) there were more downregulated genes than upregulated genes, suggesting 18G7H6A3 has an inhibitory effect on gene expression. Detailed analyses identified several differentially expressed genes including FZDB, FZD7, WNT7B, FBW11, FZD1, DVL1, CSNK2A1 and CTNNB1.

In cervical cancer, there may be a close correlation between LGR5 expression and CTNNB1. In other studies, over-expression (using LGR5 recombinant vector) or dowregulation of LGR5 (using shRNA) resulted in upregulation or downregulation of CTNNB1, respectively (Chen Q, Cao HZ, Zheng PS. 2014. Oncotarget 5: 9092-105). Additionally, analysis of immunohistochemical slides from cervical cancer patients showed a significant correlation between LGR5 and CTNNB1 expression. In this study, CTNNB1 expression was investigated further using QPCR (to measure transcript level) and Western Blotting (to assess protein expression). Using human specific primers, CTNNB1 expression was investigated in pancreatic and CRC tumors. Similar to LGR5 expression explained in Example 45, treatment with SOC increased CTNNB1 expression and the combination of 18G7H6A3 and SOC resulted in a reduction in CTNNB1 expression. Additionally, CTNNB1 expression was reduced about 35% in CT1 tumors treated with 18G7H6A3. Thus, treatment with 18G7H6A3 inhibits CTNNB1. Expression of β-catenin and phospho-β-catenin (indicative of lack of activity in Wnt pathway) was investigated by western blot analysis. Western blot data in ASPC1 tumors confirmed QPCR data in which 18G7H6A3 as single agent or in combination with SOC upregulated pβ-catenin suggesting inhibition of Wnt pathway activity in these tumors (FIG. 16).

Other components of the Wnt pathway including p-β-catenin, GSK-3β (total and phospho), and LRP6 were investigated in a series of CRC, pancreatic and breast tumors. Quantification of Western blot data showed significant inhibition of Wnt pathway signaling in ASPC1 and PANC1 tumors but also revealed some trends in favor of Wnt pathway downregulation in other models. BMCRC086 tumors that were not responsive to treatment with 18G7H6A3 were also negative for the expression of LGR5 and Wnt signaling pathway components, further supporting that the mechanism of action for 18G7H6A3 was specifically targeting LGR5 and inhibiting Wnt signaling.

Expression of Wnt pathway genes in pancreatic tumors including ASPC1, PANC1 and JH109 was investigated. Based on in vivo data, in both PANC1 and ASPC1 there was a difference in tumor volume between 18G7H6A3- vs. PBS-treated tumors. In contrast, JH109 tumors did not respond to a standard treatment regimen with either 18G7H6A3 single agent or SOC chemo combination. Differences in Wnt gene expression in responsive cells (PANC1 and ASPC1) and non-responsive cells (JH109) were investigated. In combo treated groups, Wnt6, FZD8, FOSL1, Wnt11, NFATC and FZD5 were downregulated in both ASPC1 and PANC1 combo-treated tumors, are were upregulated in JH109 tumors. In both the pancreatic and CRC data, genes including WNT11, WNT6, FRZB and PRICKEL were downregulated in PANC1, ASPC1, CT1 and CT3 cells, but not in JH109 cells.

Gene Tree analysis identified potential genes co-regulated in pancreatic tumors treated with 18G7H6A3 that included Wnt11, FRAT1, LEF1, GSK3B, FZD8 and LRP6. Analysis of differentially expressed transcripts in each treatment also identified genes that were up/down regulated more than 2 fold in pancreatic tumors (FIG. 17). Some genes, such as Wnt7A, were common between all the tumors in 18G7H6A3 vs. control treated tumors.

Example 41—18G7H6A3 Inhibits Transcription in CT1 Tumors

Expression of 18G7H6A3-targeted genes were investigated in early vs. late tumorigenesis. Mice were implanted mice with CT1, and tumors were harvested from control, 18G7H6A3, FOLFIRI or combo groups at days 3, 10 and 17. Total RNA from each tumor at day-3 was harvested and prepared for gene array hybridization using Illumina human chips. Overall analysis of differentially expressed genes (more than 1.5 or 2 folds, p<0.05) showed that in tumors treated with 18G7H6A3 (as single agent or in combination with FOLFIRI) there are more downregulated genes than upregulated ones. This suggested that treatment with 18G7H6A3 may have had a more suppressive impact on overall cellular transcriptional machinery. PCA (Principal Component Analyses) also showed a proximity in overall gene expression in 18G7H6A3 and control treated tumors. However, when 18G7H6A3 was added to FOLFIRI (i.e. combo group) there was clear separation between combo vs. FOLFIRI suggesting that targeting LGR5 may have significantly changed gene expression in FOLFIRI-treated tumors.

Analysis of differentially expressed genes in 18G7H6A3 vs. Vehicle identified several tumor promoters such as ANGPT2, AKAP12 and ADM that were downregulated in 18G7H6A3 treated tumors, and also several tumor suppressors such as DAB1, MIR655, NKX1-2 that were upregulated in 18G7H6A3 treated tumors (FIG. 18). Conversely FOLFIRI treatment appears to upregulate tumor promoters (FBN2, HKDC1, ABCB1, FGF2) and also some tumor suppressors such as TRIB3, ATF3 and TIMP3 (FIG. 19). Combination of FOLFIRI and 18G7H6A3 resulted in downregulation of more tumor promoters such as ALDOC, CDH5, ITGA2 and also upregulation of more tumor suppressors such as ZBTB11, ITPKA, PSMC3IP and BAK1 (FIG. 20).

Example 42—18G7H6A3 Treatment Significantly Reduces Human CTCs in Peripheral Blood in Orthotopic Models of Pancreatic Patient Derived Xenografts

To investigate the role of 18G7H6A3 in inhibition of primary tumor growth and metastasis, LGR5 expression was examined in a series of pancreatic patient derived xenograft samples, and PANC1424 cells and PANC1427 cells.

Tumor samples were subcutaneously implanted in NOD/SCID (non-obese diabetic severe combined immunodeficient) mice and subsequently implanted into the pancreas in recipients designated for in vivo studies. Tumor volume was measured weekly in ultrasound and mice with tumors ˜100 mm³ were enrolled into the efficacy study and were treated with the followings: 1—MOPC isotype (15 mg/kg twice/week; ip); 2—18G7H6A3 (15 mg/kg twice/week; ip); 3—SOC (Gemzar 50 mg/kg; ip twice per week and Abraxane 30 mg/kg iv twice per week); 4—Combination of 18G7H6A3 and SOC at the above doses. At the end of the study, peripheral blood from each tumor bearing mouse was collected for CTC (using flow cytometry) and circulating DNA assessments. For flow cytometry, blood samples were treated with RBC lysis buffer (ACK buffer, Life Tech, CA) using manufacturer protocol and were stained with human HLA-FITC (eBiosciences, CA) and human LGR5-AF647 (BD Pharmingen, CA) for 30 min at 4° C. Cells were washed with staining buffer (PBS-FBS 3%) twice and 7AAD (7-aminoactinomycin) prior to acquisition in the FACS calibur machine in the laboratory and the data were analyzed using FCS Express software (De Novo, Calif.).

LGR5 was expressed in various pancreatic patient derived xenograft samples. Human CTCs were detected in the peripheral blood. While percentage of HLA+ cells did not significantly change in MOPC vs. 18G7H6A3, the percentage of circulating HLA+LGR5+ cells was significantly reduced in 18G7H6A3 treated mice (FIG. 21).

The percentage of HLA+ cells did not significantly change in chemo vs. combo treated mice, however, combination of 18G7H6A3 and SOC almost completely ablated HLA+LGR5+ cells in both concurrent and debulk settings (FIG. 22A, and FIG. 22B). 18G7H6A3 treatment (as single agent or in combination with SOC) significantly reduces human CTCs in peripheral blood in orthotopic models of pancreatic patient derives xenografts.

Example 43—LGR5 Expression in Other Models

LGR5 expression was investigated in skin samples from Cynomolgus macaques (Cynos) using flow cytometry and RNAscope. Skin samples from Cynos were treated with vehicle or various doses of 18G7H6A3 (G2:10 mg/kg; G3:50 mg/kg; and G4: 150 mg/kg) at day 0, 7, 14 and 21. At study termination, skin samples were provided in DMEM supplemented with antibiotic (penicillin and streptomycin) and antimycotic solution (Anti-Anti 100×, Life Technologies, CA). Skin samples were digested using a cocktail of collagenases and thermolysin (Liberase, Roch Inc, CA). Skin progenitors (SPs) were isolated after overnight incubation with Liberase and mechanical disruption. SPs were stained with Rat anti-human LGR5 (AF647, BD Pharmingen, CA) and were analyzed in a calibur machine in the laboratory. Data analyses using FCS Express (Denovo Software, CA) showed that LGR5 was detectable in Cynos SPs, however, there was no significant difference in LGR5 frequency between 18G7H6A3 (at different doses) vs. vehicle treated group. Using RNAscope, LGR5 was detectable in skin areas especially in hair follicles and to a much lesser extent in skin epithelial cells. There was no significant difference in LGR5 positive area in vehicle vs. 18G7H6A3 treated samples.

Gene expression peripheral blood monocytes isolated from the Cynos was investigated. Total RNA was extracted using Qiagen RNeasy kit and cDNA was synthesized using Superscript cDNA Synthesis Kit (Life Technologies, CA). The cDNA from each treatment was pooled and was added to RT² Sybergreen qPCR master mix (SABiosciences, MA). The final mixture was added to each well of a 96-well plate containing Cyno QPCR primers for chemokines or inflammatory cytokines. PCR thermal profile included: 95° C. for 10 min and 40 cycles of 95° C. 15 sec and 60° C. 1 min followed by melt curve stage. Data (Ct values) in each plate was normalized by subtracting from the corresponding GAPDH and the abundance of each transcript was calculated using 2̂DCT equation. Analyses of the number of transcripts differentially expressed (more than 2 folds) between any of the 18G7H6A3 group vs. vehicle treated group showed that, consistent with gene array data, there are much more downregulated genes than the upregulated ones. With dose escalation there were less upregulated genes and more downregulated genes. The G4-recovery (G4R) group in which Cynos did not receive any treatment for 4 weeks after the last dose of 18G7H6A3 showed almost similar number of up-down-regulated genes. Detailed analysis identified differentially expressed genes (CCL11, IL3, SPP1, CCL13, CXCL6 and TNFRSF11b) whose expression was inversely correlated with 18G7H6A3 dose i.e. highest in 10 mg/kg and lowest in 150 mg/kg.

Genes that were commonly downregulated between the treatments included CCL1, IFNγ, CCR8, IL2, IL3 and IL4, some of which are enriched in M1 or M2 macrophages.

Example 44—Inhibition of Small Cell Lung Cancer Tumor Growth In Vivo by a Humanized Anti-LGR5 Antibody

Patient derived small cell lung cancer xenograft model. Dissociated tumor cells from BLG293 tumors were implanted into CB.17 SCID mice in Matrigel subcutaneously, and monitored twice weekly for tumor size and body weight. When tumors reached an average of 130 mm3, mice were randomized. Mice were treated with either PBS, antibody control MOPC, or 18G7H6A3. Mice were dosed BIW at 15 mg/kg for. All mice were monitored twice weekly for body weight and tumor size, as well as overall health and appearance, until termination.

18G7H6A3 showed significant anti-tumor activity compared to PBS (24.9% tumor growth inhibition) and MOPC antibody (24.7% tumor growth inhibition) controls.

Example 45—18G7H6A3 Increases Survival in Mice with Pancreatic Tumors that Relapse Following Debulk Chemotherapy Therapy

Panc1427 (UCSD1427) tumors were completely debulked (regressed) by treatment with chemotherapy (Gemcitabine/Abraxane) and 18G7H6A3. When tumors were regressed, chemotherapy was removed and mice were treated with either 18G7H6A3 or no treatment. Animals treated with 18G7H6A3 were noticeably more healthy compared to the control animals, where several mice had to be euthanized due to severe health observations such as lameness or body weight loss. At day 150, 7/8 mice treated with 18G7H6A3 and chemotherapy were alive, versus 4/8 mice treated with chemotherapy alone. FIG. 23 summarizes the results.

Example 46—Administration of 18G7H6A3 to Patients

A Phase I, Dose Escalation Study of BNC101 (anti-LGR5 humanized monoclonal antibody) in patients with metastatic colorectal cancer is performed as described below. Name of Finished Product: BNC101 Solution for Infusion. Name of Active Ingredient: BNC101, 18G7H6A3, ET101, LGR5 Antibody.

Study Objectives

To determine the maximum tolerated dose (MTD), recommended Phase II dose (RP2D), safety, tolerability and pharmacokinetic (PK) profile of BNC101 administered intravenously to patients with metastatic colorectal cancer. The primary objective is to determine the MTD of BNC101, both as single agent and in combination chemotherapy in metastatic colorectal cancer patients. The secondary objectives are as follows. To determine the RP2D of BNC101, both as single agent and in combination chemotherapy in metastatic colorectal cancer patients. To evaluate the safety and tolerability of BNC101 [adverse events (AEs), dose omissions or delays]. To assess for immunogenicity of BNC101 (production of antibodies against BNC101). To determine the pharmacokinetics (PK) of BNC101 (half-life, volume of distribution and clearance), both as single agent and in combination with chemotherapy. To make a preliminary assessment of the Overall Response Rate (ORR), Progression-Free Survival (PFS) and Overall Survival (OS) of metastatic colorectal cancer patients treated with BNC101. Exploratory objectives are as follows. To assess changes in disease-related biomarkers (CEA). To evaluate biomarkers of activity [pharmacodynamics, e.g. circulating tumor cells (CTCs), LGR5+ cells, circulating tumor DNA].

Safety Endpoints

To assess treatment-emergent events (clinical and laboratory data). To evaluate dose interruptions and discontinuations.

Key Patient Selection Criteria

1. Signed written Informed Consent. 2. Age >18 years. 3. Eastern Cooperative Oncology Group (ECOG) performance status score of 0-1. 4. Histologically or cytologically confirmed colorectal cancer patients who have failed at least 2 lines of chemotherapy (monotherapy treatment cohorts) or at least 1 line of chemotherapy (combination treatment cohorts) for metastatic disease, and in the opinion of both physician and patient it is not unreasonable to try experimental therapy. Adjuvant FOLFOX (Folinic acid; Fluorouracil (5-FU); and Oxaliplatin) within the last 6 months is considered a line of therapy. A maintenance strategy post 1st line treatment is not considered as an additional line of therapy. 5. Patients must have accessible tumor lesions amenable to biopsy which would not put the patient or their treatment at risk. Patients in monotherapy escalation cohort 3 and onwards, the monotherapy expansion cohort, and all combination treatment patients, agree and are willing to provide 2 serial tumor lesion biopsies (a minimum of 2 fresh cores/punches preferred whenever possible). Biopsies can be from liver metastases, in lieu of the primary tumor. The presence of tumor tissue in fresh biopsies is to be certified by a trained pathologist using appropriate extemporaneous histology or cytology procedures. 6. Measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. 7. No known brain metastases. 8. Life expectancy of at least ≥12 weeks. 9. Normal organ and marrow function: a. Absolute neutrophil count >1,500/mL without growth factor support in the past 14 days prior to enrollment. b. Platelets >100,000/mL without transfusions in the past 14 days prior to enrollment. c. Hemoglobin ≥9.0 g/dL—Patients may be transfused or receive erythropoietic treatment to meet this criterion. d. Total bilirubin <1.5× institutional upper limit of normal (ULN) (<2×ULN for subjects with Gilbert's syndrome). e. Serum Albumin ≥3 g/dL. 10. Aspartate aminotransferase (AST) (serum glutamic oxaloacetic transaminase, SGOT) and alanine aminotransferase (ALT) (serum glutamic pyruvate transaminase, SGPT)<2.5× institutional ULN (for subjects with hepatic involvement <5× institutional ULN but cannot be associated with elevated bilirubin). 11. For patients receiving biopsies, prothrombin time (PT) and activated partial thromboplastin time (APTT)/international normalized ratio (INR) within normal limits (±15%). 12. Creatinine <1.5× institutional ULN OR Creatinine clearance >60 mL/min/1.73 m2 for subjects with creatinine levels above institutional normal. 13. Adequate contraception in women and men of fertile potential.

Dosing and Schedule

Escalation will take place in two separate sets of cohorts, in a staggered fashion: single agent BNC101 dose escalation will precede dose escalation in the combination chemotherapy cohorts. In the latter, BNC101 will be dose-escalated in combination with FOLFIRI. Combination chemotherapy cohorts will not commence treatment until the RP2D has been determined in the monotherapy cohorts. Escalation in the combination chemotherapy cohorts will commence one level below the single agent RP2D of the monotherapy cohorts. Although no additive toxicities are expected from combining BNC101 with chemotherapy, two additional de-escalation levels below the initial dose will be available, should they be required.

BNC101 Monotherapy

The standard 3+3 Phase I study design will be used. The starting dose will be 1/30th of the highest no-observed-adverse-event-level (NOAEL) dose in animals (2.5 mg/kg in humans), which has been calculated taking into account species differences in receptor binding. Subsequent BNC101 dose levels will be 5, 10, and 15 mg/kg.

The schedule of administration will be weekly (q1w).

Cycle duration will be 4 weeks (28 days) (4 weekly infusions), with no week of rest between cycles.

Dose escalation will be conducted to determine the MTD. No dose escalation or reduction will be allowed within a dose cohort. In all instances, BNC101 should be administered in the morning, to enable timely preparation and shipment of blood samples for translational research on the same day.

Dose escalation will begin with a cohort of 3 patients at a dose of 2.5 mg/kg ( 1/30th of the highest NOAEL dose in animals). If, at the end of 28 days (4 administrations of BNC101), no CTCAE grade ≥2 AEs attributable to the study drug are observed, a second cohort of 3 patients will be treated at the next dose level (5 mg/kg). Further dose escalations will continue in cohorts of 3 patients (starting at 10 mg/kg and then 15 mg/kg).

If 1 of 3 subjects in a 3-subject cohort experiences a dose-limiting toxicity (DLT), that dose level will be expanded to 6 subjects. If 2 or more subjects experience a DLT, no further subjects will be dosed at that level and 3 additional subjects will be added to the preceding dose cohort unless 6 subjects have already been treated at that dose level.

Subjects will be assessed for DLTs from the time of the first dose through 28 days. Dose escalation for newly enrolled subjects, if appropriate, will occur after all subjects in a cohort have completed their Day 28 DLT assessment. Subjects with stable disease or a response at or after Day 56 (2 cycles) will be allowed to continue to receive weekly doses of BNC101 until disease progression.

If no DLTs are reported at the highest BNC101 dose tested, the cohort of patients demonstrating a PK profile with BNC101 exposure comparable to the maximum exposure demonstrated in laboratory animals (cynomolgus monkeys) will be the RP2D.

A minimum of 9 additional subjects will be further enrolled at the highest dose level that results in <2 of the 6 subjects experiencing a Grade 3 (not including a Grade 3 infusion reaction that resolves in 24 hours) or Grade 4 AE (DLT), or after adequate PK exposure has been achieved, in the absence of such toxicities.

BNC101 in Combination with FOLFIRI

After BNC101 monotherapy dose escalation is completed, declared safe and the RP2D reached, the metastatic colorectal cancer patients can be started on their combination chemotherapy cohorts with BNC101 at 1 dose level below the RP2D identified in monotherapy. These cohorts will contain 3 subjects each. Escalation will proceed as per the same rules used with monotherapy cohorts. Should this initial BNC101 dose show DLTs in 2 of a maximum of 6 patients, 2 additional de-escalation cohorts will be provided, following the 3+3 rules, to identify the RP2D in combination chemotherapy.

If no DLTs are reported at the highest BNC101 dose combination tested, the cohort of patients demonstrating a PK profile with BNC101 exposure comparable to the maximum exposure demonstrated in laboratory animals will be the RP2D in combination with chemotherapy.

A minimum of 9 additional subjects will be further enrolled in the combination cohorts at the highest dose level that results in <2 of the 6 subjects experiencing a Grade 3 (not including a Grade 3 infusion reaction that resolves in 24 hours) or Grade 4 AE (DLT) or after adequate PK exposure has been achieved, in the absence of such toxicities.

FOLFIRI components: Irinotecan (IRI)—Starting dose 180 mg/m² (over 90 minutes on Day 1) Leucovorin (LV)—Starting dose 400 mg/m² (administered over 120 minutes on Day 1 concurrently with IRI) 5-FU bolus—Starting dose 400 mg/m² (administered after LV on Day 1, then) 5-FU infusion—Starting dose 2400 mg/m² (administered over 48 hours starting on Day 1). FOLFIRI Cycles are repeated every 14 days.

DLT Definition

A DLT is defined as any of the following occurring in Cycle 1 (Days 0-28) of any given escalation cohort: Grade 3 or 4 non-hematological toxicity (including anaphylactic reactions) using the NCI CTCAE v4.0; Grade 3 nausea of more than 48 hours duration or Grade 4 vomiting or Grade ≥3 diarrhea, either occurring despite appropriate treatment; Grade 4 thrombocytopenia of any duration and Grade 4 uncomplicated neutropenia (i.e., without fever or infection) of any duration in the monotherapy cohorts or lasting >7 days in the combination chemotherapy cohorts. Grade 4 febrile neutropenia requiring hospitalization and any Grade 3 hematologic toxicity requiring treatment delay beyond 3 weeks and prolongation of QTc interval to ≥500 msec or a 60 msec increase from baseline mean QTc interval. Any other drug-related ≥ Grade 3 non-hematologic adverse event (including anaphylactic reactions), except hyperlipidemia in subjects not receiving maximum medical management or electrolyte abnormalities that may be managed with supplements. All AEs will be considered as potentially related to treatment unless there is a clear-cut relationship between the observed toxicity to disease progression (PD). Adverse events meeting these criteria will not be considered for the purposes of reporting DLTs or in determining the MTD.

Length of Treatment

Patients will be treated until PD, intolerable toxicity, withdrawal of consent, or study termination by the sponsor, whichever occurs first.

Sample Size

Up to approximately 54 patients will be treated in this study to determine the toxicity profile, DLTs, and MTD and/or RP2D of BNC101, both in monotherapy and in combination chemotherapy. It is anticipated that 1 to 6 evaluable patients per dose level cohort will provide sufficient data to assess the toxicity and PK profile of BNC101. Once the RP2D is identified, expansion of these cohorts (to at least 9 additional patients) will occur in both the monotherapy and combination chemotherapy groups. If no DLTs are reported at the highest BNC101 dose tested, the cohort of patients demonstrating a PK profile with BNC101 exposure comparable to the maximum exposure demonstrated in laboratory animals will be the RP2D.

Frequency of Visits

Every 7 (±3) days for each week of the 4-week cycle. Survival follow-up information and subsequent anti-cancer therapies will be collected every 3 months after cessation of treatment, until death, loss to follow-up, patient withdrawal of consent, or study termination by the sponsor.

Safety Assessments

Subjects will be assessed for DLT from Days 0-28 (Cycle 1). Adverse events will be reported through 30 days after the last dose. Safety will be reported continuously. Adverse events occurring between Day 28 and 30 days after the last dose will be reported, but will not be integral to the determination of the MTD. Performance status will be scored weekly, and physical examination will be conducted at baseline, at the beginning of each cycle (e.g., every 4 weeks), at the end-of-study (EOS) visit and at the time of resolution of any AEs.

Electrocardiogram (ECG) Monitoring

All 12-lead ECGs are to be obtained in triplicate and at least 5 minutes apart. ECGs will be obtained at baseline (in the 14 days previous to first dosing), on Cycle 1 Day 1 and Day 15 intensively and at pre-dose on Days 8 and 22. ECGs will be obtained pre-dose on Day 1 of Cycle 2 and subsequent cycles. An EOS ECG must be collected. ECGs will be forwarded to a local laboratory for assessment.

Response Assessments

Computed tomography (CT) scans will be performed at baseline and every 8 weeks. First response assessment will be at Day 56 (after 2 cycles). Radiologic studies for antitumor response will be repeated at the EOS visit if not done within the previous 28 days. Phone follow-up or review of records will be done on a monthly basis for 6 months and every 3 months thereafter for 18 months (total of 24 months).

Pharmacokinetic Assessment

Pharmacokinetic samples will be obtained intensively on Cycle 1, Days 1 and 15, and sparsely on Days 8 and 22. Samples will be obtained sparsely on Day 1 of Cycle 2 and subsequent cycles. Blood samples will be taken pre-dose (before the infusion is started), during the infusion, and post-infusion (after the end of the infusion) of BNC101.

Cellular and Molecular Biomarkers Assessment

Patients will have blood taken at baseline, weekly during Cycle 1, Day 1 of each subsequent cycle and at the end of treatment, to measure levels of CTCs and biomarkers (including, but not limited to LGR5) as an indicator of pharmacodynamic effect. Patients will also have 2 matched skin biopsies at baseline and Cycle 1, Day 22 (each biopsy will be 2 fresh core/punches). Patients may have additional hair samples (including collection of the hair follicle) taken.

Tumor Lesion Biopsies

No biopsies are required for monotherapy escalation cohorts 1 and 2. Matched biopsies (baseline and Day 22) are mandatory for monotherapy escalation cohort 3 and onwards, and the monotherapy expansion cohort. Matched biopsies will be mandatory for all combination treatment patients. Each biopsy will be a minimum of 2 fresh cores/punches preferred whenever possible. Biopsies can be from liver metastases, in lieu of the primary tumor. The presence of tumor tissue in fresh biopsies is to be certified by a trained pathologist using appropriate extemporaneous histology or cytology procedures. All samples will be de-identified. The identity of the individual may not be ascertained by the investigators or the sponsor. Password-protected data residing in the central reference laboratory will not be released to patients or physicians treating the patients. Patient information will be used strictly in anonymous reporting of research data. Such research data will not be placed in patient charts or made available to clinicians and will not and cannot be used for diagnostic or treatment purposes.

Immunogenicity Assessments

The presence of anti-BNC101 antibodies will be tested at baseline, prior to each dose, at treatment termination, and every 4 weeks after discontinuation of treatment for 12 weeks. Due to the different nature and expected evolution of patients in the monotherapy vs. the combination therapy cohorts, different number of samples may be obtainable in each.

The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. 

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 5. A method of treating a subject having a cancer comprising administering an effective amount of a humanized monoclonal antibody or an antigen-binding fragment thereof that specifically binds leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) to the subject in need thereof, wherein: the monoclonal antibody comprises a heavy chain comprising SEQ ID NO:13 and a light chain comprising SEQ ID NO:14; the monoclonal antibody is administered weekly for at least 4 weeks; the monoclonal antibody is administered intravenously; and the dosage of the monoclonal antibody is between about 2.5 mg/kg to about 15 mg/kg.
 6. The method of claim 5, wherein the monoclonal antibody is administered in combination with a chemotherapeutic agent.
 7. The method of claim 6, wherein the chemotherapeutic agent is selected from the group consisting of folinic acid, fluorouracil, irinotecan, gemcitabine and nanoparticle albumin-bound paclitaxel (ABRAXANE).
 8. The method of claim 7, wherein an initial dose of the monoclonal antibody is administered prior to administration of a chemotherapeutic agent.
 9. The method of claim 7, wherein the monoclonal antibody is administered in combination folinic acid, fluorouracil, and irinotecan.
 10. The method of claim 7, wherein an initial dose of the monoclonal antibody is administered prior to administration of folinic acid, fluorouracil, and irinotecan.
 11. The method of claim 7, wherein an initial dose of the irinotecan is about 180 mg/m² administered over about 90 minutes.
 12. The method of claim 11, wherein an initial dose of the folinic acid is about 400 mg/m² administered over about 120 minutes and concurrently with the initial dose of the irinotecan.
 13. The method of claim 12, wherein an initial dose of the fluorouracil is about 400 mg/m² administered after administration of the initial dose of the folinic acid.
 14. The method of claim 13, wherein the folinic acid, fluorouracil, and irinotecan are administered every 14 days.
 15. The method of claim 5, wherein the monoclonal antibody is administered in combination with an additional therapeutic agent selected from the group consisting of bevacizumab, aflibercept, cetuximab, and panitumumab.
 16. The method of claim 5, wherein the cancer comprises a solid tumor.
 17. The method of claim 5, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, pancreatic cancer, breast cancer, and lung cancer.
 18. The method of claim 5, wherein the cancer is selected from the group consisting of colon cancer comprising an APC mutation, colon cancer comprising an KRAS mutation, metastatic colorectal cancer, metastatic pancreatic cancer, triple-negative breast cancer, and small cell lung cancer.
 19. The method of claim 5, wherein the cancer is a metastatic colorectal cancer.
 20. The method of claim 5, wherein the subject has a characteristic selected from the group consisting of: failed at least 1 line of prior chemotherapy for metastatic disease prior to administration of the monoclonal antibody; has no known brain metastases; has a life expectancy of 12 weeks or more; has an absolute neutrophil count greater than about 1500 cells/mL without growth factor support in the 14 days prior to administration of the monoclonal antibody; has a platelet count greater than 100,000 platelets/mL without transfusions in the 14 days prior to administration of the monoclonal antibody; has a hemoglobin greater than or equal to 9.0 g/dL; and has serum albumin greater than or equal to 3 g/dL.
 21. The method of claim 5, wherein the subject is mammalian.
 22. The method of claim 5, wherein the subject is human.
 23. A container comprising a pharmaceutical composition comprising a dose of a humanized monoclonal antibody that specifically binds leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), and a suitable pharmaceutical carrier, wherein the dose of the monoclonal antibody is between about 2.5 mg/kg to about 15 mg/kg.
 24. The container of claim 23, wherein the pharmaceutical composition is suitable for intravenous administration.
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